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  • 1985-1989  (680)
  • 1975-1979
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  • 1988  (680)
  • Biochemistry and Biotechnology  (680)
  • 1
    ISSN: 0887-3585
    Keywords: hemocyanin ; correspondence analysis ; monoclonal antibodies ; electron microscopy ; images analysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Three epitopes have been localized by immunoelectron microscopy on subunits Aa6 of the 4 × 6-meric hemocyanin of the scorpion Androctonus australis. Soluble immunocomplexes composed of monoclonal antibodies and of native hemocyanin were purified, negatively stained with uranyle acetate by the single-layer technique, and examined under the electron microscope (EM). The molecule images were digitized, aligned, and submitted to correspondence analysis according to the method of Van Heel and Frank (Ultramicroscopy 6: 187-194, 1981). A high-precision localization of the attachment point of the Fab arm to the antigen was achieved through a careful analysis of the average images. This method easily allowed the discrimination of epitopes located in different domains (Mr 20 kDa) of the same subunit. Nonoverlapping epitopes located in the same structural domain of subunit Aa6 could be distinguished by the stain exclusion patterns of their Fab arms. The method is general and may be used for epitope mapping in any antigen producing definite EM views.
    Additional Material: 12 Ill.
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  • 2
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 3 (1988), S. 184-186 
    ISSN: 0887-3585
    Keywords: new Fe-protein ; rubredoxin ; hemerythrin ; crystals ; X-ray diffraction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A newly discovered iron-containing protein, isolated from the bacterium Desulfovibrio vulgaris (Hildenborough, NCIB 8303), has been crystallized. The molecule appears to be a dimer of mass 44kDa. This protein has iron centers with spectroscopic similarities to those in rubredoxins and in hemerythrins.The X-ray diffraction shows symmetry consistent with space group I222 or I212121. Cell parameters are a = 49.2 Å, b = 81.3 Å, c= 100.1 Å, and α, β, γ = 90°. X-ray diffraction data have been collected to 3.0 Å, and a search for useful heavy atom derivatives is in progress for the analysis of the crystal structure of this Fe-protein.
    Additional Material: 1 Ill.
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  • 3
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 3 (1988), S. 187-198 
    ISSN: 0887-3585
    Keywords: amphipathic peptide ; liposomes ; peptide ; serum apolipoproteins ; synthetic ; LCAT ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The amphipathic helical theory of Segrest and colleagues (FEBS Lett.: 38: 247-253, 1974) proposes that the lipid-binding segments of serum apolipoproteins are in an alpha helical conformation. Furthermore the helices have a hydrophobic face and a hydrophilic face with a specific distribution of positively and negatively charged residues. The importance of the pattern of the charged residues in the lipid binding and lecithin:cholesterol acyltransferase (LCAT) activation by the segments is still debated. We designed a 30-residue peptide, GALA, which in the alpha helical conformation hs a hydrophilic face composed of glutamic acid residues (Sabbarao et al.: Biochemistry 26: 2964-2972, 1987). GALA behaves like the serum apolipoproteins in its interaction with dimyristoylphospatidylcholine (DMPC) at neutral pH; the amino terminal tryptophan of GALA undergoes a blue shift in its fluorescence emission spectrum, and the circular dichroism (CD) spectrum indicates that GALA acquires alpha helical structure in the presence of DMPC. A DMPC-GALA:19/1 (molar ratio) complex can be isolated by gel-permeation chromatography. This complex has a discoidal structure with the approximate dimensions of 44-Å diameter. GALA edge thickness and a 170- to 350-Å diameter. GALA activates LCAT with DMPC but not with unsaturated phospholipids as the substrate. The apparent partition coefficient of GALA into DMPC vesicles is 100-fold larger than into egg phosphatidlylcholine vesicles. The interaction of GALA with unsaturated lipids at neutral pH is so weak that no detectable change in the spectroscopic properties of GALA or the structure of the liposomes can be detected under the conditions used here. The sequence of GALA differs from previously studied model Apo A1 peptides by the absence of positively charged residues on the hydrophilic face. This indicates that positive charges in Apo A1-like peptides are not required in order to form discoidal structures with saturated phospholipids or to activate LCAT with such lipid substrates.
    Additional Material: 6 Ill.
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  • 4
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 4 (1988), S. i 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 0887-3585
    Keywords: single-standard DNA-binding protein ; protein-nucleic acid interactions ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Using ultraviolet light, both the 33,000-dalton single-standard DNA-binding protein from T4 bacteriophage (gp32)as well as a 25,000-dalton limited trypsin cleavage product of gp32 (core gp32*) that retains high affinity for single-stranded DNA can be crosslinked to an oligodeoxynucleotide, p(dT)8. After photolysis, a single tryptic peptide crosslinked to p(dT)8 was isolated by anion-exchange high-performance liquid chromatography. Gas-phase sequencing of this modified peptide gave the following sequence: Gln-Val-Ser-Gly-(X)-Ser-Asn-Tyr-Asp-Glu-Ser-Lys, which corresponds to residues 179-190 in gp32. Based on the absence of the expected phenylthiohydantoin derivative of phenylalanine 183 at cycle 5 (X) we infer that crosslinking has occurred at this position and that phenylalanine 183 is at the interface of the gp32:P(dT)8 complex in an orientation that allows covalent bond formation with the thymine radical produced by ultraviolet irradiation.
    Additional Material: 4 Ill.
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  • 6
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 4 (1988), S. 56-62 
    ISSN: 0887-3585
    Keywords: Protein engineering ; mutagenesis ; enzyme catalysis ; conformational changes ; domain movement ; hinge bending ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Yeast 3-phosphoglycerate kinase (PGK) is a monomeric enzyme (Mr ∼ 45,000) composed of two globular domains. Each domain corresponds approximately to the amino- and carboxyterminal halves of the polypeptide chain. The carboxy-terminal end extends over the interdomain “hinge” region and packs against the amino-terminal domain. It has been proposed that domain movement, resulting in closure of the active site left, is essential for the catalytic of PGK. Large-scale conformational changes have also been postulated to explain activation of the enzyme by sulfate ions. Using site-specific mutagenesis, we have removed a 15-amino-acid carboxy-terminal fragment, in order to probe its role in the substrate- and sulfate-induced conformational changes. The truncated enzyme exhibited approximately 1% of the activity of native PGK and lost the ability to undergo sulfateinduced activation. The Km for ATP was essentially unchanged (Km = 0.23mM), whereas the Km value for 3-phosphoglycerate was increased about eightfold (Km = 3.85 mM and 0.50 mM, respectively). These results suggest that the carboxy-terminal segment is important for the mechanism of substrate- and specific-induced conformational transitions. CD spectra and sedimentation velocity measurements indicate that the carboxy-terminal peptide is essential for structural integrity of PGK. The increased susceptibility of the truncated enzyme to thermal inactivation implies that the carboxy-terminal peptide also contributes to the stability of PGK.
    Additional Material: 4 Ill.
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  • 7
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 4 (1988), S. 63-70 
    ISSN: 0887-3585
    Keywords: molecular graphics ; protein complex ; electron transfer ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A three-dimensional model of an electron-transfer complex between the tetrahemic cytochrome c3 and the ferredoxin I from the sulfatereducing bacterium Desulfovibrio desulfuricans (Norway strain) has been generated through computer graphics methods. The model is based on the known X-ray structure of the cytochrome and on a model of the ferredoxin that has been derived through computer graphics modeling and energy minimization methods, from the X-ray structure of the homologous ferredoxin from Peptococcus aerogenes. Four possible models of interaction between the two molecules were examined by bringing in close proximity each of the four hemes and the redox center (4Fe-4S) of the ferredoxin and by optimizing the ion pairs interactions. One of these models shows by far the “best” structure in terms of charges, interactions, and complementary f the topology of the contact surfaces. In this complex, the distance between the iron atoms of the ferredoxin redox center and the hemic iron atom is 11.8 Å, which compares well with those found between redox centers in other complexes. The contact surface area between the two molecules is 170 Å2.
    Additional Material: 4 Ill.
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  • 8
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 4 (1988) 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
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  • 9
    ISSN: 0887-3585
    Keywords: dUTPase ; nucleotide binding enzyme ; X-ray crystallography ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Deoxyuridine triphosphate nucleotidohydrolase (dUTPase), an enzyme in the nucleotide metabolism that is a pyrophosphatase hydrolyzing dUTP, has been crystallized. The crystals belong to the trigonal space group R3 and diffract beyond 2 Å. The native dUTPase crystals and a mercury derivative are stable in the X-ray beam and are suitable for a high resolution X-ray structure analysis.
    Additional Material: 3 Ill.
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  • 10
    ISSN: 0887-3585
    Keywords: crystallography ; structure ; refinement ; sulfonamide ; thiocyanate ; mercury ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The binding of four inhibitors - mercuric ion, 3-acetoxymercuri-4-aminobenzenesulfonamide (AMS), acetazolamide (Diamox), and thiocyanate ion - to human carbonic anhydrase II (HCA II) has been studied with X-ray crystallography.The binding of mercury to HCA II at pH 7.0 has been investigated at 3.1 Å resolution. Mercuric ions are observed at both nitrogens in the His-64 ring. One of these sites is pointing toward the zinc ion. The only other binding site for mercury is at Cys-206.The binding of the two sulfonamide inhibitors AMS and Diamox, has been reinvestigated at 2.0 and 3.0 Å, respectively. Only the nitrogen of the sulfonamide group binds to the zinc ion replacing the hydroxyl ion. The sulfonamide oxygen closet to the zinc ion is 3.1 Å away. Thus the tetrahedral geometry of the zinc is retained, refuting earlier models of a pentacoordinated zinc.The structure of the thiocyanate complex has been investigated at pH 8.5 and the structure has been refined at 1.9 Å resolution using the least-squares refinement program PROLSQ. The crystallographic R factor is 17.6%. The zinc ion is pentacoordinated with the anion as well as a water molecule bound in addition to the three histidine residues. The nitrogen atom of the SCN- ion is 1.9 Å from the zinc ion but shifted 1.3 Å with respect to the hydroxyl ion in the native structure and at van der Waals' distance from the Oγl atom of Thr-199. This is due to the inability of the Oγl atom of Thr-199 to serve as a hydrogen bond donor, thus repelling the nonprotonated nitrogen. The SCN- molecule reaches into the deep end of the active site cavity where the sulfur atom has displaced the so-called “deep” water molecule of the native enzyme. The zinc-bound water molecule is 2.2 Å from the zinc ion and 2.4 Å from the SCN- nitrogen. In addition, this water is hydrogen bonded to the Oγl atom of Thr-199 and to another water molecule.We have observed that solvent and inhibitor molecules have three possible binding sites on the zinc ion and their significance for the catalysis and inhibition of HCA II will be discussed. All available crystallographic data are consistent with a proposed catalytic mechanism in which both the OH moiety and one oxygen of the substrate HCO3- ion are ligated to the zinc ion.
    Additional Material: 9 Ill.
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  • 11
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 4 (1988), S. 251-261 
    ISSN: 0887-3585
    Keywords: protein folding kinetics ; disulfide bonds ; thiol-disulfide exchange ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Two very different mechanisms of folding have been proposed from experimental studies of disulfide formation in reduced ribonuclease A. (1) A pathway in which the rate-limiting step separates fully folded protein from all other disulfide intermediates and occurs solely in three-disulfide intermediates. (2) A multiple pathway mechanism with different rate-limiting steps for each pathway. The various rate-limiting steps involve disulfide breakage, formation, and rearrangement in intermediates with one, two, three, and four protein disulfides. To distinguish between these two mechanisms, we have carried out further studies of both unfolding and refolding.Refolding of reduced ribonuclease A requires three-disulfide intermediates to accumulate; negligible refolding occurs when only the nearly random one- and two-disulfide intermediate species are populated. Therefore, no rate-limiting steps of the type postulated in mechanism (2) occur in intermediates with one and two protein disulfides. Unfolding and disulfide reduction is an all-or-none process; no disulfide intermediates accumulate to detectable to detectable levels or precede the rate-limiting step. Mechanism (2) requires that such intermediates precede the rate-limiting step and accumulate to substantial levels.The different proposal were shown not to result from the use of different solution conditions or disulfide reagents; the two sets of data are not inconsistent. Instead, the inappropriate mechanism (2) resulted from an incorrect kinetic analysis and misinterpretation of the kinetics of disulfide formation are breakage.
    Additional Material: 6 Ill.
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  • 12
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 3 (1988) 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 13
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 3 (1988), S. 60-69 
    ISSN: 0887-3585
    Keywords: coiled-coli ; alpha-helix ; antiphagocytic ; heptad ; antigenic variation ; sequence repeats ; cell wall protein ; intermediate filaments ; myosin ; tropomyosin ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: M protein is considered a virulence determinant on the streptococcal cell wall by virtue of its ability to allow the organism to resist attack by human neutrophils. The complete DNA sequence of the M6 gene from streptococcal strain D471 has allowed, for the first time, the study of the structural characteristics of the amino acid sequence of an entire M protein molecule. Predictive secondary structural analysis revealed that the majority of this fibrillar molecule exhibits strong alpha-helical potential and that, except for the ends, nonpolar residues in the central region of the molecule exhibit the 7-residue periodicity typical for coiled-coil proteins. Differences in this heptad pattern of nonpolar residues allow this central rod region to be divided into three subdomains which correlate essentially with the repeat regions A, B, and C/D in the M6 protein sequence. Alignment of the N-terminal half of the M6 sequence with PepM5, the N-terminal half of the M5 protein, revealed that 42% of the amino acids were identical. The majority of the identities were “core” nonpolar residues of the heptad periodicity which are necessary for the maintenance of the coiled coil. Thus, conservation of structure in a sequence-variable region of these molecules may be biologically significant. Results suggest that serologically different M proteins may be built according to a basic scheme: an extended central coiled-coil rod domain (which may vary in size among strains) flanked by functional end domains.
    Additional Material: 5 Ill.
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  • 14
    ISSN: 0887-3585
    Keywords: Cα coordinates ; distance matrix ; difference distance matrix ; helix axes, strand axes ; interaxial angles ; turns ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A computer program is described that produces a description of the secondary structure and supersecondary structure of a polypeptide chain using the list of alpha carbon coordinates as input. Restricting the term “secondary structure” to the conformation of contiguous segments of the chain, the program determines the initial and final residues in helices, extended strands, sharp turns, and omega loops. This is accomplished through the use of difference distance matrices. The distances in idealized models of the segments are compared with the actual structure, and the differences are evaluated for agreement within preset limits. The program assigns 90-95% of the residues in most proteins to at least one type of secondary elementIn a second step the now-defined helices and strands are idealized as straight line segments, and the axial directions and locations are compiled from the input Cα coordinate list. These data are used to check for moderate curvature in strands and helices, and the secondary structure list is corrected where necessary. The geometric relations between these line segments are then calculated and output as the first level of supersecondary structure. A maximum of six parameters are required for a complete description of the relations between each pair. Frequently a less complete description will suffice, for example just the interaxial separation and angle. Both the secondary structure and one aspect of the supersecondary structure can be displayed in a character matrix analogous to the distance matrix format. This allows a quite accurate two-dimensional display of the three-dimensional structure, and several examples are presentedA procedure for searching for arbitrary substructures in proteins using distance matrices is also described. A search for the DNA binding helix-turnhelix motif in the Protein Data Bank serves as an exampleA further abstraction of the above data can be made in the form of a metamatrix where each diagonal element represents an entire secondary segment rather than a single atom, and the off-diagonal elements contain all the parameters describing their interrelations. Such matrices can be used in a straightforward search for higher levels of supersecondary structure or used in toto as a representation of the entire tertiary structure of the polypeptide chain.
    Additional Material: 5 Ill.
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  • 15
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 3 (1988), S. 97-101 
    ISSN: 0887-3585
    Keywords: URF ; nucleotide-binding sites ; pattern descriptor ; computer search ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: In an effort to identify the structural elements essential to a given protein function a new pattern-directed inference system has been developed. It has been employed to identify a potential dinucleotide-binding domain within the human mitochondrial unidentified reading frame 6 product, thereby supporting an earlier study that this gene may encode a NADH dehydrogenase subunit.
    Additional Material: 2 Ill.
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  • 16
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 3 (1988), S. 85-96 
    ISSN: 0887-3585
    Keywords: molecular modeling ; energy minimization ; lysine/fibrin binding ; kringle structures ; plasminogen ; tissue plasminogen activator ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The Lys binding site of kringle 1 and 4 (K1 and K4) of plasminogen (PG) has been modeled on the basis of the three-dimensional structure of kringle 1 of prothrombin and 300- and 600-MHz proton nuclear magnetic resonance observations. These structures were then compared to the corresponding regions of modeled kringle 1 and 2 of tissue plasminogen activator (PA). The coordinates of the modeled structures have been refined by energy minimization in the presence and absence of ∊-aminocaproic acid ligand in order basically to remove unacceptable van der Waals contacts. The binding site is characterized by an apparent dipolar surface, the polar parts of which are separated by a hydrophobic region of highly conserved aromatic residues. Zwitterionic ligands such as Lys and ∊-aminocaproic acid form ion pair interactions with Asp55 and Asp57 located on the dipolar surface; the latter are also conserved in all the Lys binding kringles. The cationic center of the dipolar surface is Arg71, in the case of PGK4, and is composed of Arg34 and Arg71 in PGK1. The doubly charged anionic/cationic interaction centers of the latter might account for the larger binding constants of PGK1 for like-ligands but the modeling suggests that PGK4 might be kinetically faster in binding bulkier ligands. The binding site region of PAK2, which also binds Lys, resembles those of PGK1 and PGK4. Since PAK2 lacks both cationic center Arg residues, ligand carboxylate binding appears to be accomplished though an imidazolium ion of His64, which is located just below the outer surface of the kringle.
    Additional Material: 6 Ill.
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  • 17
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 3 (1988), S. 102-112 
    ISSN: 0887-3585
    Keywords: bacterial chemotaxis ; sensory adaptation ; protein modification ; membrane protein ; receptor protein ; transmembrane signalling ; Escherichia coli ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The Trg protein is one of a family of transducer proteins that mediate chemotactic response in Escherichia coli. Transducers are methylaccepting proteins that gain or lose methyl esters on specific glutamyl residues during sensory adaptation. In this study, the significance of multiple sites of methylation on transducer proteins was addressed by using oligonucleotide-directed, site-specific mutagenesis to substitute an alanyl residue at each of the five methyl-accepting sites in Trg. The resulting collection of five mutations, each inactivating a single site, was analyzed for effects on covalent modification at the remaining sites on Trg and for the ability of the altered proteins to mediate sensory adaptation. Most of the alanyl substitutions had substantial biochemical effects, enhancing or reducing methyl-accepting activity of other sites, including one case of activation of a site not methylated in wild-type protein. Analysis of the altered proteins provided explanations for many features of the complex pattern of electrophoretic forms exhibited by Trg. The mutant proteins were less efficient than normal Trg in mediating adaptation. Correlation of biochemical and behavioral data indicated that reduction in the number of methyl-accepting sites on the transducer lengthened the time required to reach an adapted state.
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  • 18
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 3 (1988), S. 113-120 
    ISSN: 0887-3585
    Keywords: evolution ; proximal histidine ; distal histidine ; heme enzyme ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Human myeloperoxidase and human thyroid peroxidase nucleotide and amino acid sequences were compared. The global similarities of the nucleotide and amino acid sequences are 46% and 44%, respectively. These similarities are most evident within the coding sequence, especially that encoding the myeloperoxidase functional subunits. These results clearly indicate that myeloperoxidase and thyroid peroxidase are members of the same gene family and diverged from a common ancestral gene. The residues at 416 in myeloperoxidase and 407 in thyroid peroxidase were estimated as possible candidates for the proximal histidine residues that link to the iron centers of the enzymes. The primary structures around these histidine residues were compared with those of other known peroxidases. The similarity in this region between the two animal peroxidases (amino acid 396-418 in thyroid peroxidase and 405-427 in myeloperoxidase) is 74%; however, those between the animal peroxidases and other yeast and plant peroxidases are not significantly high, although several conserved features have been observed. The possible location of the distal histidine residues in myeloperoxidase and thyroid peroxidase amino acid sequences are also discussed.
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  • 19
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 3 (1988), S. 130-137 
    ISSN: 0887-3585
    Keywords: peptide synthesis ; chymotrypsin specificity ; polyethylene glycol ; nonaqueous solvents ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Chymotrypsin modified with polyethylene glycol was successfully used for peptide synthesis in organic solvents. The benzene-soluble modified enzyme readily catalyzed both aminolysis of N-benzoyl-L-tyrosine p-nitroanilide and synthesis of N-benzoyl-L-tyrosine butylamide in the presence of trace amounts of water. A quantitative reaction was obtained when either hydrophobic or bulky amides of L- as well as D-amino acids were used as acceptor nucleophiles, while almost no reaction occurred with free amino acids or ester derivativesThe acceptor nucleophile specificity of modified chymotrypsin as a catalyst in the formation of both amide and peptide bonds in organic solvents was quite comparable to that in aqueous solution as well as to that of the leaving group in hydrolysis reactions. By contrast, the substrate specificity of modified chymotrypsin in organic solvents was different from that in water since arginine and lysine esters were found to be as effective as aromatic amino acids to form the acyl-enzyme with subsequent synthesis of a peptide bond.
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  • 20
    ISSN: 0887-3585
    Keywords: proton transport ; energy transduction ; purple membrane ; proton wire ; Schiff base counter-ion ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The techniques of FTIR difference spectroscopy and site-directed mutagenesis have been combined to investigate the role of individual tyrosine side chains in the proton-pumping mechanism of bacteriorhodopsin (bR). For each of the 11 possible bR mutants containing a single Tyr→Phe substitution, difference spectra have been obtained for the bR→K and bR→M photoreactions. Only the Tyr-185→Phe mutation results in the disappearance of a set of bands that were previously shown to be due to protonation of a tryosinate during the br→K photoreaction [Rothschild et al.: Proceedings of the National Academy of Sciences of the United states of America 83:347, (1986)]. The Tyr-185→Phe mutation also eliminates a set of bands in the bR→M difference spectrum associated with deprotonation of a Tyr; most of these bands (e.g., positive 1272-cm-1 peak) are completely unaffected by the other ten Tyr→Phe mutations. Thus, tyrosinate-185 gains a proton during the bR→K reaction and loses it again when M is formed. Our FTIR spectra also provide evidence that Tyr-185 interacts with the protonated Schiff base linkage of the retinal chromophore, since the negative C=NH+ stretch band shifts from 1640 cm-1 in the wild type to 1636 cm-1 in the Tyr-185→Phe mutant. A model that is consistent with these results is that Tyr-185 is normally ionized and serves as a counter-ion to the protonated Schiff base. The primary photoisomerization of the chromophore translocates the Schiff base away from Tyr-185, which raises the pKa of the latter group and results in its protonation.
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  • 21
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 3 (1988), S. 243-251 
    ISSN: 0887-3585
    Keywords: protein evolution ; structural homology ; ribosome structure ; x-ray crystallography ; common motif ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The structure of two prokaryotic ribosomal proteins, the carboxyterminal half of L7/L12 from Escherichia coli (L12CTF) and 1.30 from Bacilus Stearothermophilus display a remarkably similar fold in which alpaha-helices pack onto one side of an antiparallel, three-stranded, beta-pleated sheet. A detailed comparison of the structures by least-squares methods reveals that more than two-thirds of the alpha carbons can be superimposed with a root mean square distance of 2.33 Å. The principal difference is an extra alpha-helix in L12 CTF. The sequences of the proteins display a distinct conservation in regions which are crucial to the common fold, in particular the hydrophobic core. It is proposed that the similarity is a result of divergent evolution.
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  • 22
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 4 (1988), S. 77-88 
    ISSN: 0887-3585
    Keywords: protein structure ; diffraction ; anomalous scattering ; x-ray crystallography ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The molecular structure of lamprey hemoglobin was previously determined and refined by conventional crystallographic analysis. In this study, the structural analysis has been repeated in the course of developing the method of multiwavelength anomalous diffraction (MAD) for phase determination. New experimental and analytical procedures that were devised to perform this determination should have general applicability. These include an experimental design to optimize signal strength and reduce systematic errors, experimental evaluation of anomalous scattering factors, and a least-squares procedure for analyzing the MAD data. MAD phases for the structure at 3Å resolution are as accurate overall as the multiple isomorphous replacement (MIR) phases determined previously.
    Additional Material: 5 Ill.
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  • 23
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    Proteins: Structure, Function, and Genetics 4 (1988), S. 89-98 
    ISSN: 0887-3585
    Keywords: membrane proteins ; channels ; circular dichroism spectroscopy ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The secondary structure of alamethicin, a membrane channel-forming polypeptide, has been examined by circular dichroism spectroscopy to determine the relationship of its conformation in organic solution to its conformation in a membrane-bound state. The spectrum of alamethicin in small unilamellar dimyristoyl phosphatidylcholine vesicles is significantly different from its spectrum in 10% methanol/acetonitrile, the solvent from which it was crystallized (Fox and Richards: Nature 300:325-330, 1982), as well as its spectrum in methanol, the solvent in which NMR studies have been done (Banerjee and Chan: Biochemistry 22:3709-3713, 1983). This suggests that structural models based on studies of the molecule in organic solvents may not be entirely appropriate for the membrane-bound state. To distinguish between different models for channel formation and insertion, two different methods were used to associate the alamethicin with vesicles; in addition, the effect of oligomerization on the conformation of the membrane-bound state was investigated. These studies are consistent with a modified insertion model in which alamethicin monomers, dimers, or trimers associate with the bilayer and then spontaneously oligomerize to form a prechannel with a higher helix content. This aggregate could then “open” upon application of an appropriate gating transmembrane potential.
    Additional Material: 4 Ill.
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  • 24
    ISSN: 0887-3585
    Keywords: 2H NMR ; selective deuteration ; tryptophan internal motion ; SSI-subtilisin complex ; protein conformational equilibrium ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Deuterium NMR spectroscopy was used to study internal motions of a deuterium-labeled single tryptophan (Trp) residue (per subunit) of Streptomyces subtilisin inhibitor (SSI) in solution. The free inhibitor with the five ring protons of the Trp replaced with deuterons showed a narrow resonance component (56 Hz) of about one-quarter of the total intensity, in addition to the broad resonance component (about 600 Hz) at 25°C, showing that it exits in an equilibrium mixture of two conformers, in one of which the typtophan side chain is highly mobile. In analogy to the two structures of SSI found in the crystal, these two conformers were attributed to the one in which the contact between the α-lobe and the beta;-lobe of the subunit is tight and the other in which the same contact is loose. When SSI forms a complex with subtilisin BPN′, the broad component becomes invisibly broad, but the narrow component increases with even further narrowing, suggesting that the binding to the enzyme favors the “loose” conformer over the “tight” conformer.
    Additional Material: 4 Ill.
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  • 25
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    Proteins: Structure, Function, and Genetics 4 (1988), S. 137-147 
    ISSN: 0887-3585
    Keywords: eye lens proteins ; protein association ; crystal packing ; surface area ; homologous proteins ; point mutations ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A comparative study of intermolecular interactions in crystals of two homologous low molecular weight proteins, γ-II and γ-IIIb crystallins, from calf eye lens was carried out. Crystal packings for these proteins are very different: intermolecular contact areas compose about 33% of the total accessible surface area of γ-II as compared with 13% in γ-III. Two key residues seem to be mainly responsible for the differences in protein association in the crystal medium. These are Ser 103 and Leu 155 in γ-II, which are replaced by Met 103 and His 155 in γ-IIIb. A similar substitution of these residues is observed in different gene products of γ-crystallins from a number of vertebrates. This is consistent with the existence of a genetically controlled mechanism for determining intermolecular association of γ-crystallins in the native medium of the lens.
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  • 26
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    Proteins: Structure, Function, and Genetics 4 (1988), S. 148-156 
    ISSN: 0887-3585
    Keywords: Protein structure ; empirical energy ; energy minimization ; molecular dynamics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A method for the prediction of hydrogen positions in proteins is presented. The method is based on the knowledge of the heavy atom positions obtained, for instance, from X-ray crystallography. It employs an energy minimization limited to the environment of the hydrogen atoms bound to a common heavy atom or to a single water molecule. The method is not restricted to proteins and can be applied without modification to nonpolar hydrogens and to nucleic acids. The method has been applied to the neutron diffraction structures of trypsin ribonuclease A, and bovine pancreatic trypsin inhibitor. A comparison of the constructed and the observed hydrogen positions shows few deviations except in situations in which several energetically similar conformations are possible. Analysis of the potential energy of rotation of Lys amino and Ser, Thr, Tyr hydroxyl groups reveals that the conformations of lowest intrinsic torsion energies are statistically favored in both the crystal and the constructed structures.
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  • 27
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    Proteins: Structure, Function, and Genetics 4 (1988) 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 28
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    Proteins: Structure, Function, and Genetics 4 (1988), S. 157-164 
    ISSN: 0887-3585
    Keywords: synthetic inhibitors ; serine proteinase crystallography ; active site geometry ; computer graphics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Proteinase K, the extracellular serine endopeptidase (E.C. 3.4.21.14) from the fungus Tritirachium album limber, is homologous to the bacterial subtilisin proteases. The binding geometry of the synthetic inhibitor carbobenzoxy-Ala-Phechloromethyl Ketone to the active site of proteinase K was the first determined from a Fourier synthesis based on synchrotron X-ray diffraction data between 1.8 Å and 5.0 Å resolution. The protein inhibitor complexes was refined by restrained least-squares minimization with the data between 10.0 and 1.8 Å. The final R factor was 19.1% and the model contained 2,018 protein atoms, 28 inhibitors atoms, 125 water molecules, and two Ca2+ ions. The peptides portion of the inhibitor is bound to the active center of proteinase K by means of a three-stranded antiparallel pleated sheet, with the side chain of the phenylalanine located in the P1 site. Model building studies, with lysine replacing phenylalanine in the inhibitor, explain the relatively unspecific catalytic activity of the enzyme.
    Additional Material: 5 Ill.
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  • 29
    ISSN: 0887-3585
    Keywords: α/β barrels ; crystal structure ; glucose isomerase ; xylose isomerase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The structure of Xylose isomerase (X.I.) from Actinoplanes missouriensis has been solved to 2.8 Angstroms resolution. Phases were determined from a single Eu3+ derivative and from the noncrystallographic 22 symmetry of the tetrameric molecule. An atomic model was built and subjected to restrained crystallographic refinement. The resulting model is shown to be closely similar to the recently reported X.I.'s structures from three other bacterial sources. Each monomer is found to be composed of an eight-stranded α/β “T.I.M.” barrel forming an N-terminal domain of 328 residues followed by a large loop of 66 residues embracing an adjacent subunit. Analysis of intersubunit packing shows that the X.I. tetramer is an assembly of two tight dimers. The β barrel fits a simple hyperboloid model as other T.I.M. barrels do. The active site, identified as the binding site for the inhibitor xylitol, is located at the carboxyl end of the beta strands in the barrel next to a pair binding site for Eu3+ ions, which are assumed to the sites for the divalent ions involved in catalysis. Active sites in the tetramer are oriented towards the interface between dimmers. It is suggested that subunit interfaces might stabilize the active site region and this might explain the oligomeric nature of the other α/β barrel enzymes.
    Additional Material: 5 Ill.
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  • 30
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    Proteins: Structure, Function, and Genetics 4 (1988), S. 173-181 
    ISSN: 0887-3585
    Keywords: DNA binding protein ; ligand binding ; equilibrium dialysis ; dimer ; stability ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Availability of the three-dimensional structure of the trp repressor of Escherichia coli and a large group of repressor mutants has permitted the identification and analysis of mutants with substitutions of the amino acid residues that from the tryptophan binding pocket. Mutant aporepressors selected for study were overproduced using a multicopy expression plasmid. Equilibrium dialysis with 14C-tryptophan and purified mutant and wild type aporepressors was employed to determine tryptophan binding constants. The results obtained indicate that replacement of theronine 44 by methionine (TM44) or arginine 84 by histidine (RH84) lowers the affinity for tryptophan approximately two-and four-fold, respectively. Replacement of ariginine 54 by histidine (RH84) or glycine 85 by ariginine (GR85) results in complete loss of tryptophan binding activity. Purified mutant and wild type aporepressors were used in vitro heterodimer studies. The trp repressor of E. coli functions as a stable dimer. A large number of trp repressor mutants prduces defective repressors that are transdominant to the wild type repressor in vivo. The transdominance presumably results from the formation of inactive or slightly active heterodimers between the mutant and wild type polypeptide subunits. An in vitro assay was developed to detect and measure heterodimer formation. Heterodimer formation was thermally induced, and heterodimers were separated on nondenaturing polyacrylamide gels. Aporepressors readily formed heterodimer formation upon treatment at 65°C for 3 minutes. Heterodimer formation was significantly retarded by the presence of the corepressor, L-tryptophan. Indole-3-propionic acid, 5-methyl tryptophan, and other analogs of tryptophan, as well as indole, also inhibited heterodimer formation. These results indicate that the presence of the indole moiety in the corepressor binding pocket increases the stability of the dimer.
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  • 31
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    Proteins: Structure, Function, and Genetics 4 (1988), S. 205-210 
    ISSN: 0887-3585
    Keywords: slow-binding inhibition ; transition-state analog ; conformational change ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Boronic acid derivatives of good peptide substrate of the serine proteases cause slow-binding inhibition, manifested as biphasic binding (Kettner and Shenvi: J. Biol. Chem. 259:15106-15114, 1984). These inhibitors are thought to act as reaction-intermediate analogs. Three peptides Boronic acids - Ac-Pro-boro-Val-OH, DNS-Ala-Pro-boro-Val-OH, and Ac-Ala-Ala-Pro-boro-Val-OH - were chosen for farultraviolet circular dichroism (CD) studies in order to determine whether the second phase involves a conformational change of pancreatic elastase. The dipeptide is a simple competitive inhibitors (Ki = 0.27 μM) and the latter are slow-binding inhibitors (Ki = 16.4 and 0.25 nM, respectively). Spectral deconvolution and correction for the formation of antiparallel β-sheet by the peptide inhibitors itself indicate that there is no significant change in the secondary structure of the enzyme in the either the initial or final inhibitors complex. A kinetic experiment confirmed that the slow-binding step was not associated with a CD spectral change, and that therefore a protein conformational change was not responsible for the sow binding.
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  • 32
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    Proteins: Structure, Function, and Genetics 4 (1988) 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 33
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    Proteins: Structure, Function, and Genetics 4 (1988), S. 211-227 
    ISSN: 0887-3585
    Keywords: protein-folding ; multiple pathways ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The diffusion-collision model has been used to analyze the folding kinetics of myoglobins. The microdomains, which are the basic units that coalesce during the folding, are identified with the helices and the stabilizing contacts between helices are determined form the native structure. Both association and disassociation reactions are included and a range of stabilization parameters is investigated to determine the variations in overall rate and the relative contributions made by the different intermediates during the folding process. In a comparison of folding to the native state and to the midpoint of the folding transitions. (i.e., 50% native protein at the completion of the reaction) significant differences in the contributing intermediates are found.
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  • 34
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    Proteins: Structure, Function, and Genetics 4 (1988), S. 262-273 
    ISSN: 0887-3585
    Keywords: hierarchical assignment ; cereal grain ; mistletoe ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Methods that analyze protein circular dichroism (CD) spectra for fractions of secondary structure are evaluated for the plant protein crambin, which has a known high-resolution crystal structure. In addition, a two-step secondary structure prediction scheme is presented and used for the toxins homologous to crambin, shown by others to have secondary structures similar to crambin.The test of CD spectral analysis methods with the protein crambin employed two computer programs and several CD basis sets. Crambin's crystal structure, known to 0.945 Å resolution (Hendrickson, W.A., Teeter, M.M. Nature 290:107-113, 1981), allows accurate evaluation of results. Analysis with the protein spectra basis sets (Provencher, S. W., Glöckner, J. Biochemistry 20:33-37, 1981) as modified (Manavalan, P., Johnson, W. C., Jr. Anal. Biochem. 167:76-85, 1987) agreed most closely with crambin's crystal structure. This method was then applied to the CD spectra of the membrane-active toxins homologous to crambin (α1- and β-purothionin, phoratoxin A and B, an viscotoxin A3 and B).The new program SEQ (pronounced “seek”) was developed to assign the secondary structure along the protein chain in a hierarchical fashion and applied to the plant toxins. The method constrained the secondary structure fractions to those from CD analysis and combined standard statistical methods with amphipathic helix location.Both CD-arrived secondary structure percentages and sequence assignment indicate that the viscotoxins are structurally most similar to crambin. Purothionin's secondary structure was predicted to be fundamentally similar to crambin's with a difference at the start of the first helix. This assignment agreed with Raman and NMR analyses of Purothionin and lends validity to the method presented here. Differences from the NMR in the CD secondary structure fraction analysis for phoratoxin suggest interference in the CD from tryptophan residues.
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  • 35
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    Proteins: Structure, Function, and Genetics 4 (1988), S. 274-282 
    ISSN: 0887-3585
    Keywords: crystallography ; refinement ; structure ; carbonic anhydrase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The structure of human erythrocytic carbonic anhydrase II has been refined by constrained and restrained structure-factor least-squares refinement at 2.0 Å resolution. The conventional crystallographic R value is 17.3%. Of 167 solvent molecules associated with the protein, four are buried and stabilize secondary structure elements. The zinc ion is ligated to three histidyl residues and one water molecule in a nearly tetrahedral geometry. In addition to the zinc-bound water, seven more water molecules are identified in the active site. Assuming that Glu-106 is deprotonated at pH 8.5, some of the hydrogen bond donor-acceptor relations in the active site can be assigned and are described here in detail. The Oγ1 atom of Thr-199 donates its proton to the Oε1 atom of Glu-106 and can function as a hydrogen bond acceptor only in additional hydrogen bonds.
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  • 36
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    Proteins: Structure, Function, and Genetics 4 (1988), S. 294-295 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Additional Material: 3 Ill.
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  • 37
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    Proteins: Structure, Function, and Genetics 3 (1988) 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 38
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    Proteins: Structure, Function, and Genetics 3 (1988), S. 1-17 
    ISSN: 0887-3585
    Keywords: computer modeling ; trifluoperazine ; conformational change ; calcium binding proteins ; hydrophobic binding interactions ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Among the known regulatory proteins that are conformationally sensitive to the binding of calcium ions, calmodulin and troponin-C have the greatest primary sequence homology. This observation has led to the conclusion that the most accurate predicted molecular model of calmodulin would be based on the X-ray crystallographic coordinates of the highly refined structure of turkey skeletal troponin-C. This paper describes the structure of calmodulin built from such a premise. The resulting molecular model was subjected to conjugate gradient energy minimization to remove unacceptable intramolecular non-bonded contacts. In the analysis of the resulting structure, many features of calmodulin, including the detailed conformation of the Ca2+-binding loops, the amino- and carboxy-terminal hydrophobic patches of the Ca2+-bound form, and the several clusters of acidic residues can be reconciled with much of the previously published solution data. Calmodulin in missing the N-terminal helix characteristic of troponin-C. The deletion of three residues from the central helical linker (denoted D/E in troponin-D) shortens the molecule and changes the orientation of the two domains of calmodulin by 60° relative to those in troponin-C. The molecular model has been used to derive two possible binding sites for the antipsychotic drug trifluoperazine, a potent competitive inhibitor of calmodulin activity.
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  • 39
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    Proteins: Structure, Function, and Genetics 3 (1988), S. 18-31 
    ISSN: 0887-3585
    Keywords: flexibility ; trp repressor ; DNA-binding domains ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: An orthorhombic crystal form of trp repressor (aporepressor plus L-tryptophan ligand) was solved by molecular replacement, refined to 1.65 Å resolution, and compared to the structure of the repressor in trigonal crystals. Even though these two crystal forms of repressor were grown under identical conditions, the refined structures have distinctly different conformations of the DNA-binding domains. Unlike the repressor/aporepressor structural transition, the conformational shift is not caused by the binding or loss of the L-tryptophan ligand. We conclude that while L-tryptophan binding is essential for forming a specific complex with trp operator DNA, the corepressor ligand does not lock the repressor into a single conformation that is complementary to the operator. This flexibility may be required by the various binding modes proposed for trp repressor in its search for and adherence to its three different operator sites.
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  • 40
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    Proteins: Structure, Function, and Genetics 3 (1988), S. 53-59 
    ISSN: 0887-3585
    Keywords: ricin ; retroviral integrase ; conserved residues ; homologous sequences ; active site ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Plant ribosome-inhibiting proteins are shown to be homologous at the domain level to RNase H form Escherichia coli and to two regions of the pol gene product of retroviral reverse transcriptases. One of these regions carries the viral integrase or int function, while the other has previously been suggested to contain the viral RNase H exo activity. Several residues conserved among the ribosome inhibitors, E. coli RNase H, and the integrase proteins are seen to occupy a prominent cleft in the tertiary structure of the ribosome inhibitor ricin, suggesting roles in binding or catalysis. It is likely that these homologous sequences represent modern derivatives of an ancient protein-folding unit capable of nucleic acid binding and modification which has been incorporated into a variety of enzyme functions.
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  • 41
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    Proteins: Structure, Function, and Genetics 3 (1988), S. 32-52 
    ISSN: 0887-3585
    Keywords: alpha-helix ; distance-dependent ; finite-difference method ; Poisson-Boltzmann equation ; protein electrostatics ; rhodanese ; solvent effects ; subtilisin ; Tanford-Kirkwood theory ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Electrostatic interactions between pairs of atoms in proteins are calculated with a model based on the linearized Poisson-Boltzmann equation. The equation is solved accurately by a method that takes into account the detailed shape of the protein. This paper presents applications to several systems. Experimental data for the interaction of ionized residues with an active site histidine in subtilisin BPN' allow the model to be tested, using various assumptions for the electrical properties of the protein and solvent. The electrostatic stabilization of the active site thiolate or rhodanese is analyzed, with attention to the influence of α-helices. Finally, relationships between electrostatic potential and charge-charge distance are reported for large and small globular proteins. The above results are compared with those of simpler electrostatic models, including Coulomb's law with both a distance-dependent dielectric constant (∊ = R) and a fixed dielectric constant (∊ = 2), and Tanford-Kirkwood theory. The primary conclusions are as follows: (1) The Poisson-Boltzmann model agrees with the subtilisin data over a range of ionic strengths; (2) two α-helices generate a large potential in the active site of rhodanese; (3) ∊ = R overestimates weak electrostatic interactions but yields relatively good results for strong ones; (4) Tanford-Kirkwood theory is a useful approximation to detailed solutions of the linearized Poisson-Boltzmann equation in globular proteins; and (5) the modified Tanford-Kirkwood theory overscreens the measured electrostatic interactions in subtilisin.
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  • 42
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    Proteins: Structure, Function, and Genetics 3 (1988), S. 199-207 
    ISSN: 0887-3585
    Keywords: folding pathway ; hydrophobic interaction ; long-range and long-distance interactions ; secondary structure ; tertiary structure ; module structure ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: To explain the rapidity of the process of protein folding, we cite two aspects of hydrophobic interaction: its long-range nature and the specificity of pairing after the formation of secondary structures. These two factors, when incorporated with the growth-type mechanism, can determine the folding pathway of proteins. This mechanism is applied to myoglobin. Appropriate introduction of side chins of amino acid residues and the heme group attached to His 93 yield a refolded tertiary structure that is in good agreement with the native structure.
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  • 43
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    Proteins: Structure, Function, and Genetics 3 (1988) 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 44
    ISSN: 0887-3585
    Keywords: distance-restrained molecular dynamics ; 2D NOE-spectroscopy ; tertiary structure ; solution conformations ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The technique of two-dimensional nuclear magnetic resonance (2D-NMR) has recently assumed an active role in obtaining information on structures of polypeptides, small proteins, sugars, and DNA fragments in solution. In order to generate spatial structures from the atom-atom distance information obtained by the NMR method, different procedures have been developed. Here we introduce a combined procedure of distance geometry (DG) and molecular dynamics (MD) calculations for generating 3D structures that are consistent with the NMR data set and have reasonable internal energies. We report the application of the combined procedure on the lac repressor DNA binding domain (headpiece) using a set of 169 NOE and 17 “hydrogen bond” distance constraints. Eight of ten structures generated by the distance geometry algorithm were refined within 10 ps MD simulation time to structures with low internal energies that satisfied the distance constraints.Although the combination of DG and MD was designed to combine the good sampling properties of the DG algorithm with an efficient method of lowering the internal energy of the molecule, we found that the MD algorithm contributes significantly to the sampling as well.
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  • 45
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    Proteins: Structure, Function, and Genetics 3 (1988), S. 230-242 
    ISSN: 0887-3585
    Keywords: melittin ; spin-labelling ; EPR spectroscopy ; membrane-protein interaction ; protein-protein interaction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Spin-labeled derivatives of the bee venom protein, melittin, were obtained by reacting on the average one of the four amino groups of the protein with succinimidyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-3-carboxylate All 16 statistically possible reaction products with 0, 1, 2, 3 or 4 spin labels per protein were then separated in a single pass with reversed phase high performance liquid chromatography. With the help of trypsin digestion and diode array detection it was possible to assign the primary structure of all 16 eluting fractions. All fractions with only one spin label per protein were purified for electron paramagnetic resonance measurement. The labeling sites cover different regions of the protein: one is at the N-terminus, one at lysine-7, and two are near the C-terminus at lysine-21 and lysine-23, respectively. This set of specifically labeled melittins was used to study the structure and dynamics of melittin in aqueous solutions and when bound to neutral or negatively charged membranes. In aqueous solution a reduction in rotational correlation time and appearance of spin-spin interaction was observed during salt-induced transition from a random coil monomer to a mostly α-helical retramer. Membrane binding to phospholipid bilayers in low or high ionic strength was reflected only in a further decrease in mobility. The absence of any spin interaction in the membrane-bound state suggests that melittin is monomeric under these conditions. All derivatives were able to detect these structural changes, but melittin labeled at the N-terminal amino group was especially valuable. Because of postulated intramolecular hydrogen bonding, this label reflects directly the motion of the entire protein or tetramer. Broadening experiments with chromium oxalate show that all labeled sites are at least partially exposed to the aqueous phase when melittin is bound to membranes. This suggests that an α-helical melittin monomer binds to membranes with its axis parallel to the membrane surface.
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  • 46
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    Proteins: Structure, Function, and Genetics 3 (1988), S. 252-255 
    ISSN: 0887-3585
    Keywords: streptomyces malayensis ; antitumor antibiotic ; holoprotein antibiotic ; crystallization of mitomalcin ; amino acid composition ; partial amino acid sequence ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The antitumor antibiotic protein mitomalcin, from the microorganism Streptomyces malayensis, has been purified to apparent homogenity and crystallized. The crystals belong to space group P212121 and have the following cell parameters : a=27.2 Å, b=34.1 Å, c=101.7 Å, and alpha;=β=γ=90°. These crystal properties are extremely similar to crystals of the antitumor protein neocarzinostatin (11.7 kilodaltons [kDa]) from Streptomyces carzinostaticus in spite of differing pH conditions for crystallizing the two proteins and an apparent difference in molecular weight is similar to that of neocarzinostatin. An amino acid composition analysis of mitomalcin indicates that some differences may exist between the two molecules, but a preliminary amino acid sequence analysis of the first 37 residues found no difference in the N-terminal region of the molecule.
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  • 47
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    Proteins: Structure, Function, and Genetics 3 (1988), S. 256-261 
    ISSN: 0887-3585
    Keywords: zymogen activation ; protein engineering ; autoproteolytic processing ; protein structure/function ; renaturation ; rDNA expression ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Site-specific mutagenesis of the gene encoding bovine prochymosin was used to produce a mutated zymogen in which seven contiguous amino acids of the N-terminal propeptide had been deleted and an eighth residue had been substituted. This altered region spans the normal site of autocatalytic proteolysis that occurs at the same time as (enzymatic) activation of prochymosin at acidic pH. Activation of the mutated zymogen at pH 4.5 was extremely slow, and cleavage occurred at an unusual Ser-Lys bond in the prochymosin incubated at pH 2 generated the usual pseudochymosin by cleavage of the normal Phe-Leu bond, but at a rate severalfold slower than the authentic zymogen. These results indicate that even after deletion of seven of 42 amino acids of the propeptide the mutant protein could still assume a prochymosin (zymogen) structure, although these changes did result in striking differences in acid-catalyzed activation and processing reactions at one but not the other of the two processing sites of prochymosin.
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  • 48
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    Proteins: Structure, Function, and Genetics 4 (1988) 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 49
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    Proteins: Structure, Function, and Genetics 3 (1988), S. 262-265 
    ISSN: 0887-3585
    Keywords: protein conformation ; conformational equilibria ; free energy stimulation ; protein folding ; thermodynamic perturbation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A direct attack on the protein-folding problem has been initiated with the free energy perturbation methods of molecular dynamics. The complete conformational probability map for the alanine dipeptide is presented. This work uses the SPC model for the explicit hydration of the dipeptide. Free energy differences for the four observed minima (β, αR, αL, C7ax) are given, and the free energy barriers between minima are outlined.
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  • 50
    ISSN: 0887-3585
    Keywords: consensus sequences ; secondary structure ; mannose 6-phosphate ; substrate specificity ; proteolytic processing ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Recently developed computer programs, including secondary structure and epitopic site predictions, have been used to align lysosomal proteins for maximum homology, based on conservative interchanges, and the aligned sequences have been searched for potential sites for posttranslational modification, glycosyaltion, and binding and catalysis of substrate. The homology and prediction of the posttranslational modification of the α- and β-subunits of hexosaminidase is in good agreement with previous observations, and an explanation of the differing substrate specificities of the two subunits is advanced. We shows that the striking homology between α-glucosidase and isomaltase is reflected in that apparent conversation of the active site in both enzymes. Nonhomologous regions have been examined in detail in a search for binding sited for glycogen and maltose, and two such sites have been tentatively identified. A highly redundant consensus sequence for the Phosphorylation of mannose in lysosomal proteins, YXX(Y, W, or F), is suggested.
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  • 51
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    Proteins: Structure, Function, and Genetics 4 (1988), S. 190-204 
    ISSN: 0887-3585
    Keywords: computer graphics ; energy minimization ; proteolytic enzymes ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Two genes that are expressed when precursor cytotoxic T lymphocytes are transformed to T killer cells have been cloned and sequenced. The derived amino acid sequences, coding for the cytotoxic cell protease 1 (CCP1) and Hannuka factor (HF) are highly homologous to members of the serine proteinase family. Comparative molecular model building using the known three-dimensional structures and the derived amino acid sequences of the lymphocyte enzymes has been provided useful information, especially in predicting the conformations of the substrate binding sites. In applying this modelling procedure, we used the X-ray structures of four serine proteinase to provide a structurally based sequences alignment: α-chymotrypsin (CHT), bovine trypsin (BT), Streptomyces griseus trypsin (SGT), and rat must cell protease 2 (RMCP2). The root mean square differences in α-carbon atom positions among these four structures when compared in a pairwise fashions range form 0.79 to 0.97 Å for structurally equivalent residues. Te sequences of the two lymphocyte enzymes were then aligned to these proteinase using chemical criteria and the superimposed X-ray structures as guides. The alignment showed that the sequence of CCP1 was most similar to RMCP2, whereas HF has regions of homology with both RMCP2 and BT. RmCP2 and BT as templates for HF, the molecular models were constructed. Intermolecular steric clashes that resulted from replacement of amino acid chains of the templates by the aligned residues of CCP1 and HF were relieved by adjustment of the side chain conformational angels in an interactive computer graphics device. This process was followed by energy minimization of the enzyme model to optimize the stereochemical geometry and to relieve any remaining unacceptably close nonbonded contacts. The resulting model of CCP1 has an arginie residue at position 226 in the specificity pocket, thereby predicting a substrate preference for P1 aspartate or glutamate residues. The model also predicts favorable binding for a small hydrophobic residue at the P2 position of the substrate. The primary specificity pocket of HF resembles that of BT and therefore predicts a lysine or arginine preference for the P1 residue. The arginine at position 99 in the model of HF suggests a preference for aspartate or glutamate side chains in the P2 positions of the substrate. Both CCP1 and HF have a free cysteine in the segment of the polypeptide 88 to 93. Models of the dimeric form of these enzymes can be constructed by forming disulfide bridges between the suitably oriented monomers, ie., Cys88-Cys88′ for CCP1 and Cys93-Cys93′ for HF.
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  • 52
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    Proteins: Structure, Function, and Genetics 4 (1988), S. 229-239 
    ISSN: 0887-3585
    Keywords: repressor proteins ; helix dipole ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The DNA-binding helix pairs in gene repressor and activator were compared with other approximately perpendicular pairs of adjacent helices in the known protein structures. Two other examples of closely matching conformations were found in cytochrome c peroxidase (residues 153-174) and in ribosomal L7/L12 protein (residues 68-89). Another group of such offset “lap-joints” are the Ca-binding “EF hand” structures, which bind a positive rather than a negative ligand. The EF hands turn out to match the DNA-binding motifs quite well (outside of the loop) if their sequence direction is reversed. This conformation is thus not as unusual as had been thought, but may have a more generalized role in ion binding and occasionally occur in a purely structural role.
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  • 53
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    Proteins: Structure, Function, and Genetics 4 (1988), S. 240-250 
    ISSN: 0887-3585
    Keywords: protein structure ; X-Ray crystal structure ; homology ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Two new globin structures have recently been determined at high resolution: the globin from the mollusc Aplysia limacina at 1.6 A resolution and a new refinement of the structure from sea lamprey. Two amino acid sequences of these homologous molecules have only 30% residue identity in an optimal alignment. We discuss some of the problems arising in the alignment of Aplysia globin with other globins of known structure, a challenging problem because of the distant relationship. Four independent approaches were applied to the alignment of the Aplysia and lamprey globins, including those based on individual sequence comparisons, structural analysis, and the relatively new method of templates or fingerprints derived for an entire family of proteins. We also compare these two new structures with what is already known about the globin family. A detailed description of the two structures shows that the two molecules contain the main structural features common to all the globins so far studied with several minor but interesting hitherto unobserved variations.
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  • 54
    ISSN: 0887-3585
    Keywords: bioactivity ; SK-hep-1 hepatoma ; interleukin-1 ; recombinant protein ; crystals ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The gene for human interleukin-1β was cloned from SK-hep-1 hepatoma cellular RNA and expressed at high levels in Escherichia coli both as the naturally processed form (rIL-1β) and as a variant with an additional sequence of three amino acids on the N-terminus (rIL-1β+). Expressed protein was purified to homogeneity by a sequence of steps, which included low pH incubation, adsorption and desorption from Procion Red Sepharose, sizing on a Superose 12 fast-performance liquid chromatography (FPLC) column, and anion exchange chromatography on QAE Sepharose. The final step provided a biologically active protein that migrates on twodimensional (2-D) gels as a single spot with a pI of 6.7 ± 0.2 and a molecular mass of 17,500 daltons. Concentrated solutions of rIL-1β have produced crystals by ammonium sulfate precipitation. The crystals are tetragonal, show the symmetry of space group P41 or its enantiomer, have lattice constants of a = 58.46 (1) and c = 77.02 (3) A, and scatter to at least 2 Å resolution. A structure determination based on these crystals is under way.
    Additional Material: 6 Ill.
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  • 55
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    Proteins: Structure, Function, and Genetics 3 (1988) 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 56
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    Proteins: Structure, Function, and Genetics 3 (1988), S. 139-145 
    ISSN: 0887-3585
    Keywords: Protein structure ; complement ; anaphylatoxins ; two-dimensional NMR ; computer modeling ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The model structure previously proposed for human C5a, based upon the crystal structure of the homologous protein human C3a, is compared to the solution structure of human C5a recently determined by nuclear magnetic resonance (NMR) methods in our laboratory. The general folding and helix topography of the C5a protein were modeled very well. The N-terminus, which is disordered in teh C3a crystal, was correctly predicted in the C5a model both as to its being a helix and as to its docking site on the rest of the molecule. On the other hand, the NMR data show that the biologically important C-terminal residues are disordered in solution, unlike the model and the C3a crystal structure where this region was helical.
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  • 57
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    Proteins: Structure, Function, and Genetics 3 (1988), S. 146-154 
    ISSN: 0887-3585
    Keywords: Pseudomonas toxin ; x-ray crystallography ; ADP-ribosyl transferase ; sequence homology ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Pseudomonas aeruginosa exotoxin A is representative of a class of enzymes, the monoADP-ribosyl, which catalyze the covalent transfer of an ADP-ribose moiety of NAD+ to a target substrate. Availability of the three-dimensional structure of exotoxin A provides the opportunity for mapping substrate binding sites and suggesting which amino acid residues may be involved in catalysis. Data from several sources have been combined to develop a proposal for the NAD+ binding site of exotoxin A: the binding of NAD+ fragments adenosine, AMP, and ADP have been delineated crystallographically to 6.0, 6.0, and 2.7 Å, respectively; significant sequence homology spanning 60 residues has been found between exotoxin A and diphtheria toxin, which has the identical enzymatic activity; iodination of exotoxin A, under conditions in which only tyrosine 481 is iodinated in the enzymatic domain, abolishes ADP-ribosyl transferase activity.
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  • 58
    ISSN: 0887-3585
    Keywords: monoclonal antibodies ; high-affinity combining sites ; MPD ; Effects of fluorescein binding ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: An antigen-binding fragment (Fab) from a murine monoclonal antibody (4-4-20) with high affinity for fluorescein was cocrystallized with ligand in polyethylene glycol (PEG) and 2-methl-2,4-pentanediol (MPD) in forms suitable for X-ray analyses. In MPD the affinity of the intact antibody for fluorescein was 300 times lower than the value (3.4 × 1010 M-1) obtained in aqueous buffers. This decreased affinity was manifested by the partial release of bound fluorescein when MPD was added to solutions of liganded Feb during crystallization trials, In PEG, the ligand remained firmly bound to the protein. The liganded Feb crystallized in the monoclinic space group P21 in PEG, with a = 58.6, b = 97.2, c = 44.5 Å and β = 95.2°. In MPD the space group was triclinic P1, with a = 58.3, b = 43.4, c = 42.3 Å, α = 83.9°, β = 87.6°, and γ = 84.5°. X-ray diffraction data were collected for both forms to 2.5-Å resolution. Surprisingly, the triclinic form of the liganed antifluorescyl Feb had the same space group, closely similar cell dimensions, and practically the same orientation in the unit cell as an unliganded Fab (BV04-01) with activity against single-stranded DNA.
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  • 59
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    Proteins: Structure, Function, and Genetics 4 (1988), S. 7-18 
    ISSN: 0887-3585
    Keywords: protein electrostatics ; conformational energy ; solvation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: In this report we describe an accurate numerical method for calculating the total electrostatic energy of molecules of arbitrary shape and charge distribution, accounting for both Coulombic and solvent polarization terms. In addition to the solvation energies of individual molecules, the method can be used to calculate the electrostatic energy associated with conformational changes in proteins as well as changes in solvation energy that accompany the binding of charged substrates. The validity of the method is examined by calculating the hydration energies of acetate, methyl ammonium, ammonium, and methanol. The method is then used to study the relationship between the depth of a charge within a protein and its interaction with the solvent. Calculations of the relative electrostatic energies of crystal and misfolded conformations of Themiste dyscritum hemerythrin and the VL domain of an antibody are also presented. The results indicate that electrostatic charge-solvent interactions strongly favor the crystal structures. More generally, it is found that charge-solvent interactions, which are frequently neglected in protein structure analysis, can make large contributions to the total energy of a macromolecular system.
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  • 60
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    Proteins: Structure, Function, and Genetics 4 (1988), S. 19-30 
    ISSN: 0887-3585
    Keywords: Conformation search ; CONGEN ; misfolded structures ; solvent-modified potentials ; protein folding ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Various theoretical concepts, such as free energy potentials, electrostatic interaction potentials, atomic packing, solvent-exposed surface, and surface charge distribution between native proteins and misfolded protein models. Misfolded models were constructed by introducing incorrect side chains onto polypeptide backbones: side chains of the α-helical hemerythrin were modeled on the β-sheeted backbone of immunoglobulin VL domain, whereas those of the VL domain were similarly modeled on he hemerythrin backbone. CONGEN, a conformational space sampling program, was used to construct the side chains, in contrast to the previous work,1 where incorrect side chains were modeled in all trans conformations. Capability of the conformational search procedure to reproduce native conformations was gauged first by rebuilding (the correct) side chains in hemerythrin and the VL domain: constructs with r.m.s differences from the x-ray side chains 2.2-2.4 Å were produced, and many calculated conformations matched the native ones quite well. Incorrectly folded models were then constructed by the same conformational protocol applied to incorrect amino acid sequences. All CONGEN constructs, both correctly and incorrectly folded, were characterized by exceptionally small molecular surfaces and low potential energies. Surface charge density, atomic packing, and Coulomb formula-based electrostatic interactions of the misfolded structures and the correctly folded proteins were similar, and therefore of little criteria clearly favored the native structures over the misfolded ones: (1) solvent-exposed side-chain nonpolar surface, (2) number of buried ionizable groups, and (3) empirical free energy functions that incorporate solvent effects.
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  • 61
    ISSN: 0887-3585
    Keywords: protein simulation ; dihydrofolate reductase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A study of the binding of the antibacterial agent trimethoprim to Escherichia coli dihydrofolate reductase was carried out using energy minimization techniques with both a full, all-atom valence force field and a united atom force field. Convergence criteria ensured that no significant structural or energetic changes would occur with further minimization. Root-mean-square (RMS) deviations of both minimized structures with the experimental structure with the experimental structure were calculated for selected regions of the protein. In the active site, the all-atom minimized structure fit the experimental structure much better than did the united atom structure. To ascertain what constitutes a good fit, the RMS deviations between crystal structures of the same enzyme either from different species or in different crystal environments were compared. The differences between the active site of all-atom minimized structure and the experimental structure are similar to differences observed between crystal structures of the same protein.Finally, the energetics of ligand binding were analyzed for the all-atom minimized coordinates. Strain energy induced in the ligand, the corresponding entropy loss due to shifts in harmonic frequencies, and the role of specific residues in ligand binding were examined. Water molecules, even those not in direct contact with the ligand, were found to have significant interaction energies with the ligand. Thus, the inclusion of at least one shell of waters may be vital for accurate simulations of enzyme complexes.
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  • 62
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    Proteins: Structure, Function, and Genetics 4 (1988), S. 48-55 
    ISSN: 0887-3585
    Keywords: nuclear magnetic resonance ; computer modeling ; linear peptides ; omega loop ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The solution structure of a 38-amino-acid-residue, biologically active fragment of bovine growth hormone (bGH96-133) was investigated with a combined nuclear magnetic resonance (NMR) and computer modeling approach. With the distance geometry program DISGEO and distance constraints derived from the nuclear Overhauser enhancement (NOE) experiments, it was found that residues Ser-100 to Tyr-110 circumscribe an Ω-loop, a recently categorized feature of nonregular secondary protein structure.
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  • 63
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    Proteins: Structure, Function, and Genetics 4 (1988), S. 99-122 
    ISSN: 0887-3585
    Keywords: protein structure ; protein coding regions ; sequence homology ; reading frame ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: We have examined oligopeptides with lengths ranging from 2 to 11 residues in protein sequences that show no obvious evolutionary relationship. All sequences in the Protein Identification Resource database were carefully classified by sensitive homology searches into superfamilies to obtain unbiased oligopeptide counts. The results, contrary to previous studies, show clear prejudices in protein sequences. The oligopeptide preferences were used to help decide the significance of sequence homologies and to improve the more general methods for detecting protein coding regions within nucleotide sequences.
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  • 64
    ISSN: 0887-3585
    Keywords: NMR spectroscopy ; protein dynamics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Solid-state and solution 15N nuclear magnetic resonance experiments on uniformly and specifically 15N labeled coat protein in phospholipid bilayers and in detergent micelles are used to describe the dynamics of the membrane-bound form of the protein. The residues in the N- and C-terminal portions of the coat protein in both phospholipid bilayers and in detergent micelles are mobile, while those in the hydrophobic midsection are immobile. There is evidence for a gradient of mobility in the C-terminal region of the coat protein in micelles; at 25°C only the last two residues are mobile on the 109-Hz timescale, while the last six to eight residues appear to be mobile on slower timescales and highly mobile at higher temperatures. Since all of the C-terminal residues are immobile in the virus particles, the mobility of these residues in the membrane-bound form of the protein may be important for the formation of protein-DNA interactions in the assembly process.
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  • 65
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    Biotechnology and Bioengineering 31 (1988), S. 1-10 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The kinetics of anaerobic degradation of a molasses wastewater were measured under constant pH conditions in a laboratory scale packed bed reactor. In continuous and batch experiments the formation and degradation rates of the organic acids (butyric, propionic and acetic) have been followed. The influence of hydrogen gas on the acid degradation rates has been measured and, contrary to the literature and the thermo-dynamic calculations, no inhibition was detected, biofilm diffusional effects may be the reason. Two dynamic simulation models were tested, a heterogeneous model, which considered the biofilm diffusion-reaction phenomena and a quasihomogeneous model with the same kinetics. Except for hydrogen, the diffusion effects were found to be negligible. Otherwise both models gave essentially the same results and the time profiles of acids, hydrogen, carbon dioxide and methane agreed relatively well with dynamic startup experiments. Batch experiments showed the acid concentrations to be highly sensitive to the initial molasses concentration. This aspect was not included in the model but is being investigated further.
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  • 66
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    Biotechnology and Bioengineering 31 (1988), S. 35-40 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The attrition bioreactor (ABR) combines wet ball milling and enzymatic hydrolysis in one process step. It was found that the ABR did not accelerate enzyme deacti-vation. Interfacial forces, not shear forces, caused the most deactivation. Elimination of the air-liquid interface by covering the reactor substantially increased enzyme stability. A simple exponential kinetic model was tested to predict the cellulose conversion in an ABR. Kinetic parameters were estimated from batch runs performed at various enzyme and substrate concentrations.
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  • 67
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    Biotechnology and Bioengineering 31 (1988), S. 62-70 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A model was developed and evaluated as a tool for predicting the formation of soluble products from staged thermochemical treatment of lignocellulosic materials under acidic conditions typical of autohydrolysis. The model was used to predict the general trend of hemi-cellulose and cellulose hydrolysis between pH 2 and 4 and temperatures of 170-230°C, and results were compared with experimental data. When the model was evaluated for this range of temperatures and pH values, results indicated: (1) a relatively low temperature (175°C) during the first stage allows hydrolysis of the hemi-cellulose polysaccharides without significant mono-saccharide decomposition, (2) subsequent stages at higher temperatures (equal or greater than 200°C) are needed for significant cellulose hydrolysis, but glucose decomposition will also occur, and, (3) a pH in the range of 2-2.5 will enhance polysaccharide hydrolysis while limiting monosaccharide decomposition. The model's predictions, indicating that the formation of biodegradable products could be optimized using Pretreatments at pH 2-2.5 for the pH range evaluated, were confirmed in experiments with white fir as a representative lig nocellulose.
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  • 68
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    Biotechnology and Bioengineering 31 (1988), S. 102-116 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A three-pool growth model of an individual Escherichia coli cell is described herein. The model is based on a previously developed chemically structured complex single cell growth model. The reduction in model complexity and the identification of the essential modes of motion, over the time scale of growth, is achieved by temporal decomposition and analysis of hierarchy in relaxation times. The three-pool model faithfully simulates the changes in cell size, cell shape, cell macromolecular composition, DNA initiation and termination periods, and the dependence of cell growth under abiotic glucose limitation. The predictions made by the reduced model compare favorably with both the experimental data and those of the full single cell model (SCM) without any parameter adjustments. The three-pool model has very few significant parameters and has the potential to find immediate practical use in bioreactor design and process control strategies. The model development illustrates the use of modal analysis to yield reduced physiologically realistic dynamic model of complex microbial system such as E. coll.
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  • 69
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    Biotechnology and Bioengineering 31 (1988), S. 125-129 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The regulation and kinetics of formation of an inducible, cell-bound oxacillin-hydrolyzing β-lactamase (M-OXA) by a methanol-grown Pseudomonas strain were investigated in batch, chemostat, and two-stage continuous cultures. The extent to which enzyme production occurs declines at increased growth rates, and the rate of M-OXA β-lactamase production follows a kinetic pattern that is partially growth independent and partially inversely growth linked. Growth and enzyme formation are regulated differently by medium constituents. The initial steps of M-OXA β-lactamase synthesis takes place during the exponential growth phase, and active enzyme is produced by induced nonproliferating cells, probably through a turnover mechanism.
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  • 70
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    Biotechnology and Bioengineering 31 (1988), S. 168-172 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Mouse-human hybridoma 4H11 cells producing anti-Pseudomonas sp. monoclonal antibody (IgA) grew in a serum-free medium supplemented with insulin, transferrin, ethanolamine, and selenite (ITES). The hybridoma could be applied to high-density culture in a serum-free medium supplemented with ITES, 0.5% BSA, egg yolk VLDL, and artificial blood FC-43 in a culture vessel equipped with hollow-fiber modules for medium exchange. Total cell density reached 1.1 × 107 cells/mL (viable cell density was 7.6 × 106 cells/mL), and the IgA productivity was around 20 μg/106 cells/day in the serum-free medium, which corresponded to the levels in serum-supplemented medium.
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  • 71
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    Biotechnology and Bioengineering 31 (1988), S. 208-214 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Porcine pancreatic and Chromobacterium viscosum lipases catalyze transesterification reactions between a number of sugar alcohols and various plant and animal oils in dry pyridine. The products of this process have been identified as primary monoesters of sugar alcohols and fatty acids. These enzymatically prepared sugar alcohol esters have been found to be excellent surfactants in terms of their ability to reduce interfacial and surface tensions and to stabilize emulsions.
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  • 72
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    Biotechnology and Bioengineering 31 (1988), S. 224-234 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Optimal control theory was applied to the process of batch beer fermentation. The performance functional considered was a weighted sum of maximum ethanol production and minimum time. Calculations were based on the model of Engasser et al. modified to include temperature effects. Model parameters were determined from isothermal batch fermentations. The fermentor cooling duty was the single available control. Temperature state variable constraints as well as control variable constraints were considered. The optimal control law is shown to be bang-bang control with the existence of a singular arc corresponding to isothermal operation at the maximum temperature constraint. An iterative algorithm is presented for computing appropriate switching times using a penalty-function-augmented performance functional.
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  • 73
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    Biotechnology and Bioengineering 31 (1988), S. 235-239 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A mathematical model is presented for a microporous hollow-fiber membrane extractive fermentor (HFEF). The model is based on the continuous flow of the aqueous nutrient phase and cells through the shell space of the fermentor where the fermentation reaction occurs. The product diffuses from the shell space through the hollow-fiber membrane where it is continuously removed by solvent flowing concurrently through the fiber lumen. Results for ethanol production show that the HFEF has a volumetric productivity significantly higher than that possible using conventional methods. The model predicts the existence of an optimum volume fraction of hollow fibers in the fermentor that maximizes the total volumetric productivity. This optimum is the result of a classic trade-off between the volume fraction of the fermentor required for fermentation and that required for efficient removal of the ethanol product to minimize product inhibition.
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  • 74
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    Biotechnology and Bioengineering 31 (1988), S. 278-281 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Additional Material: 1 Ill.
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  • 75
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    Biotechnology and Bioengineering 31 (1988), S. 287-294 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A simple adaptive control algorithm, for which theoretical stability and convergence properties had been previously demonstrated, has been successfully implemented on a biomethanation pilot reactor. The methane digester, operated in the CSTR mode was submitted to a shock load, and successfully computer controlled during the subsequent transitory state.
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  • 76
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    Biotechnology and Bioengineering 31 (1988), S. 311-320 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The objective of this work was to relate macroscopically measurable on-line fermentation parameters such as dissolved oxygen, off-gas oxygen and carbon dioxide, and cell mass, to the controlled production of key intracellular enzymes under carbon limited conditions. Both batch and perturbed batch aerobic fermentations were performed using two different strains of Escherichia coli, with glucose and lactose as the sole carbon sources. The two strains differed from each other only in the lac operon region of their genome. The parent strain, E. coli 3000, was inducible for the enzyme β-galactosidase. The other strain, E. coli 3300, was a constitutive mutant in the production of β-galactosidase. In all experiments, off-line assays of sugars and β-galactosidase activity were performed. It was observed that there is a clear relationship between the macroscopic on-line measurements, dissolved oxygen tension, carbon dioxide evolution rate and oxygen uptake rate, and the microscopic control phenomena of catabolite repression, catabolite inhibition, and inducer repression.
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  • 77
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    Biotechnology and Bioengineering 31 (1988), S. 345-348 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Needle-punch polyester was shown to be an effective support material for the immobilization of Trichoderma reesei Rut C30. When used as a resident inoculum for a batch process, the immobilized Trichoderma was very stable and resulted in a reduced rate of biomass generation in the bulk liquid phase as compared to cultures inoculated with free mycelium. Fed-batch fermentations with the immobilized Trichoderma produced ca. 80% of the activity of those using free cells; however, the activity was more stable and the crude enzyme broth produced had a greatly reduced biomass concentration.
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  • 78
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    Biotechnology and Bioengineering 31 (1988), S. 502-506 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Additional Material: 3 Tab.
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  • 79
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    Biotechnology and Bioengineering 31 (1988), S. 511-515 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Liquid membrane bilayers, generated by bacteriorhodopsin on a supporting membrane, exhibit photo osmosis. The phenomenon has been shown to be a consequence of light-induced electrical potential differences which develop across the liquid membrane bilayer due to the light-driven proton pumping action of bacteriorhodopsin. The variations of photo osmotic velocity with wavelength, intensity of light, and proton acceptor concentrations has been studied.
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  • 80
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    Biotechnology and Bioengineering 31 (1988), S. 536-546 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The unbound bilirubin concentration and the enzymatic rate of bilirubin degradation by bilirubin oxidase in bilirubin-serum albumin solutions have been investigated experimentally and theoretically. A stoichiometric bilirubin-serum albumin binding analysis shows that the unbound bilirubin concentration depends only on the molar ratio of the total bilirubin concentration to the total serum albumin concentration. From the theoretical analysis and the measured unbound bilirubin concentrations, serum albumin may be modelled as a molecule having two binding sites, primary and secondary, with stoichiometric equilibrium constants of K1 = 6 × 107M-1 and K2 = 4.5 × 106M-1, respectively. The rate of total bilirubin degradation in bilirubin-serum albumin mixtures is zero order. An immobilized bilirubin oxidase reactor model, which shows good agreement with experimental bilirubin conversions, is presented. At a flow rate of 1 mL/min with a 8-mL reactor volume, a 50% bilirubin conversion per pass was observed with an inlet bilirubin concentration of 350μM and a serum albumin concentration of 500μM.
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  • 81
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    Biotechnology and Bioengineering 32 (1988), S. 527-537 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The switching characteristics of a monocyclic enzyme system, in which two enzymes share substrates or co-factors in a cyclic manner, such as, → X1 + B + E1 ⇄ A + E1 + X2 →, → X3 + A + E2 ⇄ B + E2 + X4 → (E1, E2 are enzymes, X1, X3 are substrates, X2, X4 are products, A, B are cofactors), were demonstrated using computer simulations. The detailed mathematical models of biochemically possible cyclic enzyme systems were built up and the effects of rate constants and the effects of initial concentrations of enzymes and cofactors on switching characteristics were discussed. The cyclic enzyme system could function as a switching circuit when the initial concentrations of enzymes or cofactors are over a certain threshold value. Based on the present results, we further discuss the dynamic characteristics of a biochemical reactor system (bioreactor) involving this cyclic enzyme system as a switching controller.
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  • 82
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    Biotechnology and Bioengineering 32 (1988), S. 545-553 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Inactive cells of Rhizopus arrhizus have been immobilized into the form of particles of desirable particle size using a proprietary immobilization technique. The immobilized biomass particles are porous and are members of a new generation of biological origin adsorbents. The uranium adsorptive behavior of the biosorbent particles was modeled using a batch reactor mass transfer kinetic model of the biosorption process. The model successfully predicts the batch reactor adsorbate (uranium) concentration profiles and has provided significant insights on the way biosorbents function.
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  • 83
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    Biotechnology and Bioengineering 32 (1988), S. 538-544 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The concurrent production of acids and solvents and the production of acetone during continuous culture in a product-limited chemostat indicated that the culture contained a mixture of acid- and solvent-producing cells. Periodic oscillations in the yield of end products and the specific growth rate of the culture were ob served during undisturbed continuous culture at a constant dilution rate. The increased specific growth rate was associated with an increased acid yield and an increase in the rate of cell division and the proportion of short rods. The decreased specific growth rate was as sociated with an increase in the solvent yield and a decrease in the rate of cell division, resulting in the production of elongated rods. It is proposed that the oscillatory behavior observed during continuous culture is an inherent characteristic related to the shift from primary to secondary metabolism. A major consequence of the oscillation of the specific rates of growth and division in cultures containing acid- and solvent-producing cells is that it precludes the attainment of a true steady state during continuous culture.
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  • 84
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    Biotechnology and Bioengineering 32 (1988), S. 554-563 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The ideal derivatized support for the clinical use of an immobilized enzyme system should irreversibly bind active enzyme. We have investigated the behavior of heparinase and bilirubin oxidase immobilized via cyanogen bromide, tresyl chloride, epoxide, or carbodiimidazole activated natural and synthetic matrices. The protein bound to each activated support was 90% for cyanogen bromide (CNBr) activated agarose, 50-80% for tresyl chloride activated agarose, and 50% for oxirane activated acrylic (Eupergit C). The activity retention of immobilized heparinase was greatest (50%) with CNBr activated agarose while for the immobilization of bilirubin oxidase, the activity retention was greatest (25-30%) with tresyl chloride activated agarose and oxirane activated acrylic.The stability of the different covalent bonds was studied in vitro with radioiodinated enzymes. The leaching profiles showed the same trends for each support and chemistry. A plateau in portein leaching was reached after a few hours of incubatttion and the transient leaching period was well represented byu a logarithimic function of time. The amount of enzyme released from the least stable support (CNBr activated agarose) in 24 h was injected intravenously in New Zealand white rabbits. Using an indirect enzyme-linked immunnosorbant assay (ELISA), no immune responce was detected. The transient leaching profile was shortenend by washingthe enzyme-support conjugate with 1M hydroxylamine, pH8.5 intermolecular cross-linking with glutaraldehyde also improves the enzyme-support stability. Tresyl chloride and oxirane activated supports produce bonds with improved stability without adversely affecting enzymatic activity.
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  • 85
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    Biotechnology and Bioengineering 32 (1988), S. 595-603 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Cross-linked polyurethane (PU) was prepared for entrapping thermolysin. Using the immobilized thermolysin (IT), Z-L-aspartic acid (ZA) was reacted with -Lphenylalanine methyl ester (L-PM) in water-saturated ethyl acetate to give only α-Z-L-aspartylL-phenylalanine methyl ester (α-ZAPM). Ninety-four percent conversion of α-ZAPM was obtained for 30 h of reaction at 40°C when 46 mg of enzyme was entrapped. PU support prepared from polypropylene glycol (#2000) showed better properties than from polypropylene (#1000) and polyethylene (#1000). Addition of polyol could increase the gel fraction of PU. The IT PU-ll-G-3, prepared from 1/2 mole ratio of PPG (#2000)/glycerin, gave the highest gel fraction and best swelling, and 89.0% of residual activity was obtained after four times of reuse (72 h). The stability of immobilized thermolysin was good; the activity loss resulting from degradatin and leak of enzyme in each time of reuse were found only about 2%. The kinetics of immobilized thermolysin-catalyzed condensation reaction of ZA with L-PM in water-saturated ethyl acetate was found to be first order in L-PM and the Lineweaver-Burk plot of 1/V against 1/[ZA] yields a straight line, showing that the reaction involves consecutive reactions of ZA and L-PM with the immobilized enzyme and with the ZA-immobilized enzyme complex, with the second reaction being the rate determining step.
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  • 86
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    Biotechnology and Bioengineering 31 (1988), S. 240-249 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A model is developed and used to predict the dynamic behavior of the elution stage of biospecific adsorption (affinity chromatography) in a finite bath. Both nonselective and selective elution of monovalent adsorbates is considered. The model expressions account for film and pore diffusion resistances for the adsorbate(s) and the eluent, and various rate expressions for the desorption of the adsorbate from the adsorbate-ligand complex are constructed and studied. The results indicate that the duration of the elution stage depends significantly on the Sherwood number of the adsorbate and the rate of the interaction step between the ligand and the adsorbate relative to the diffusion of the adsorbate in the pore during elution. In nonselective elution, when the value of the effective pore diffusivity of the eluent is significantly larger than that of the adsorbate, the results suggest that it would be advantageous to use an initial eluent concentration in the finite bath that is only slightly higher than the critical eluent concentration in order to minimize the risk of product and ligand damage. In selective elution the amount of adsorbate recovered in the elution stage is greatly influenced by the initial concentration of the eluent and the equilibrium dissociation constants of the adsorbate-ligand and adsorbate-eluent complexes.
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  • 87
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    Biotechnology and Bioengineering 31 (1988) 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
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  • 88
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    Biotechnology and Bioengineering 31 (1988), S. 304-310 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A segregated model of multicopy plasmid propagation has been formulated which incorporates plasmid replication and partition functions, as well as the effect of plasmid presence on host growth rate. Growth of plasmid-free cells in selective medium is explicitly analyzed. The model parameters can be determined from experimentally measurable quantities. Propagation of a recombinant multicopy plasmid in the yeast Saccharomyces cerevisiae is analyzed using this model.
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  • 89
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    Biotechnology and Bioengineering 31 (1988), S. 328-335 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Batch experiments were conducted to examine the effects of several substrate analogs on the degradation of pentachlorophenol by an enrichment culture of pentachlorophenol-utilizing bacteria. The presence of substrate analogs which were unable to serve as a carbon source for growth of the culture (e.g., 3,5,6,-trichloro-2-pyridinol, 2,4-dichlorophenoxyacetic acid) decreased the rate of pentachlorophenol degradation. The presence of a utilizable substrate analog (e.g., phenol, 2,4,5-trichlorophenol) also inhibited the initial rate of pentachlorophenol degradation; however, the overall removal rate was accelerated due to an increase in cell mass concentration as a result of simultaneous growth on both substrates. These effects were shown to be predicted by a mathematical model based on a modified Monod equation. Kinetic parameters obtained from the results of laboratory studies can be used for further process analysis to define the optimal conditions for the biological treatment of complex mixtures of phenolic compounds.
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  • 90
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    Biotechnology and Bioengineering 31 (1988), S. 336-344 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Photobioreactor design and operation are discussed in terms of mixing, carbon utilization, and the accumulation of photosynthetically produced oxygen. The open raceway pond is the primary type of reactor considered; however small diameter (1-5 cm) horizontal glass tubular reactors are compared to ponds in several respects. These are representative of the diversity in photobioreactor design: low capital cost, open systems and high capital cost, closed systems. Two 100-m2 raceways were operated to provide input data and to validate analytical results. With a planktonic Chlorella sp., no significant difference in productivity was noted between one pond mixed at 30 cm/s and another mixed from 1 to 30 cm/s. Thus, power consumption or CO2 outgassing limits maximal mixing velocities. Mixing power inputs measured in 100-m2 ponds agreed fairly well with those calculated by the use of Manning's equation. A typically configured tubular reactor flowing full (1 cm diameter, 30 cm/s) consumes 10 times as much energy as a typical pond (20 cm deep flowing at 20 cm/s). Tubular reactors that flow only partially full would be limited by large hydraulic head losses to very short sections (as little as 2 m length at 30 cm/s flow) or very low flow velocities. Open ponds have greater CO2 storage capacity than tubular reactors because of their greater culture volume per square meter (100-300 L/m2 vs. 8-40 L/m2 for 1-5-cm tubes). However, after recarbonation, open ponds tend to desorb CO2 to the atmosphere. Thus ponds must be operated at higher pH and lower alkalinity than would be possible with tubular reactors if cost of carbon is a constraint. The mass transfer coefficient, KL, for CO2 release through the surface of a 100-m2 pond was determined to be 0.10 m/h. Oxygen buildup would be a serious problem with any enclosed reactor, especially small-diameter tubes. At maximal rates of photosynthesis, a 1-cm tubular reactor would accumulate 8-10 mg O2/L/min. This may result in concentrations of oxygen reaching 100 mg/L, even with very frequent gas exchange. In an open pond, dissolved oxygen rises much more slowly as a consequence of the much greater volume per unit surface area and the outgassing of oxygen to the atmosphere. The maximum concentration of dissolved oxygen is typically 25-40 mg/L. The major advantage of enclosed reactors lies in the potential for aseptic operation, a product value which justifies the expense. For most products of algal mass cultivation, open ponds are the only feasible photobioreactor design capable of meeting the economic and operating requirements of such systems, provided desirable species can be maintained.
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  • 91
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    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
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  • 92
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    Biotechnology and Bioengineering 32 (1988), S. 803-812 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Substrate and energy costs of the production of exocellular enzymes from glucose and citrate by B. Iicheniformis S1684 as well as molar growth yields corrected for these costs of product formation were calculated using data from chemostat experiments. The calculations showed that 1.46-1.73 mol glucose and 2.31-2.77 mol citrate are needed for formation and excretion of 1 mol protein. Consequently, the values of the maximal product yield from substrate (Ypsm' g/mol) are 80 〈 Ypsm 〈 95 when product is formed from glucose and 50 〈 Ypsm 〈 60 when product is formed from citrate. The higher substrate costs for product formation from citrate are due to a higher level of CO2 production during protein formation and a higher substrate requirement for the energy supply of product formation and excretion than when product is formed from glucose. The theoretical ATP requirement for protein synthesis could be determined reasonably well, but the energy costs of protein excretion could not be determined exactly. The energy costs of protein formation are higher than those of biomass formation or protein excretion. Molar growth yields corrected for the substrate costs of product formation were high, indicating a high efficiency of growth.Growth and production parameters were determined as well from experimental data of recycling fermentor experiments using a parameter optimization procedure based on a mathematical model describing biomass growth as a linear function of the substrate consumption rate and the rate of product formation as a linear function of biomass growth rate. The fitting procedure yielded two growth and production domains during glucose limitation. In the first domain the values for the maximal growth yield and maintenance coefficient were in agreement with those found in chemostat experiments at corresponding values of Yspm. Domain 2 could be described best with linear growth and product formation. In domain 2 the rate of product formation decreased and more substrate became available for biomass formation. As a consequence the specific growth rate increased in the shift from domain 1 to 2. Domain 2 behavior most probably is caused by the rel-status of B. Iicheniformis S1684.
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  • 93
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    Biotechnology and Bioengineering 32 (1988), S. 853-865 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The adsorption of cellulase from Trichoderma reesei MCG 77 on Avicel was measured at varying cellulase (2-8 g/L) and Avicel (10-200 g/L) concentrations at pH 4.8 and 50°C. Different mathematical equations were derived for the evaluation of the experimental data. The fraction of cellulase protein that can maximally be adsorbed is 0.96, and 1 g Avicel can bind maximally 0.092 g cellulase protein. The Michaelis constant for the adsorption equilibrium [cellulase] + [Avicel] ⇌ [cellulase Avicel] complex is between 2.0 and 2.3 · 10-5 mol/L. This value is based on the assumption that cellulase has an average molecular weight of 48.000. The apparent molecular weight of Avicel, i.e., that amount in grams that can bind 1 mol cellulase, is 520,000. Under maximum binding the enzyme covers on Avicel a surface of 42 m2/g, and the occupied volume is 0.186 cm3/g Avicel.
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  • 94
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    Biotechnology and Bioengineering 32 (1988), S. 873-885 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A review of the various literature data for large-scale algae production costs is described. Costs were updated and recomputed in order to compare the different schemes. Total production costs of a nonprocessed biomass range from US$0.15 to US$4.0 kg-1, according to various authors. Process performance hypotheses and proposed technologies are analyzed to explain these variations. A cost analysis for a tubular bioreactor system is then presented that shows that, assuming a productivity of 60 tons/ha yr, production costs would range from FF24 to FF29 kg-1 for such a system. Operating costs as well as fixed charges account for approximately 50% of the cost. Parametric sensitivity of these costs is then analyzed: If productivity would be 30, 45, or 90 tons/ha yr, total cost would be around FF48, FF33 and FF19 kg-1. Advantages and disadvantages of the proposed tubular technology are finally discussed.
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  • 95
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    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A chimeric plasmid (pYT760-ADH1) containing the yeast killer toxin-immunity cDNA was transformed into a leucine-histidine mutant (AH22) and into four industrial toxin-sensitive yeasts. The chimeric plasmid was very stable and expressed toxin production (89.5 ± 4.8% killer cells) in two of the transformed yeasts that contained the 2μ plasmid, but was lost within 10 generations from two other transformed pickle yeasts that did not contain the 2μ plasmid. It suggested that plasmid stability was dependent on the presence of the 2μ plasmid which is naturally present in some yeasts. The plasmid was extremely stable (100% killer cells) and expressed more toxin in the mutant strain AH22. The effects of dilution rate, D(h-1) on plasmid stability and toxin expression were studied in transformed AH22 (AH22/T3) and Montrachet 522 (522/T1) wine yeast grown in glucose-limited chemostat cultures. The results show that killer toxin production by AH22/T3 cells increased as a function of D(h-1) and that plasmid stability reached 100% at D ≥ 0.09 ± 0.01 h-1. However, with Montrachet 522/T1 transformed cells, 100% plasmid stability was seen at D ≥ 0.18 ± 0.02. h-1. We also challenged the AH22/T3 in chemostat culture (D = 0.25 h-1) with an equal number of untransformed cells (AH22). Transformed cells dominated the population (100%) within 8-10 h of growth, a time equivalent to two mean residence time.
    Additional Material: 5 Ill.
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  • 96
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 31 (1988), S. 805-820 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In order to develop a general methodology for evaluation of the gene expression efficiency for gene product, theoretical and experimental studies were undertaken using a recombinant Escherichia coli K12ΔH1Δtrp/ pPLc23trpA1 as a “gene-host cell” model system in a two-stage continuous-culture system. For this, a genetically structured kinetic model proposed earlier for biosynthesis of gene product in batch cultivation was extended to the two-stage continuous-culture system. A partial list of key parameters of the model includes the rate of plasmid segregation, specific growth rate of recombinant cell, plasmid content, rates of transcription and translation, and other parameters related to product biosynthesis. The dynamics of heterogeneous cell population containing plasmid-harboring and plasmid-free cells were also studied. Theoretical analysis of cell population dynamics shows that the recombinant cells could be maintained stably for a prolonged time in a two-stage continuous-culture system. Fermentation performance of the recombinant E. Coli cells in a two-stage continuous bioreactor system was examined experimentally, and the gene expression efficiency of a cloned gene product was determined based on the genetically structured kinetic model proposed. Based on our experimental results, the gene expression efficiency of the model gene-host cell system was found to be about twofold more efficient (i. e., 41.8 mg TrpA protein/mg plasmid DNA) as compared to the average rate of protein biosynthesis by E. coli cells. The performance of two-stage recombinant fermentation was also simulated using a mathematical model developed. General trends obtained from the model simulation agree reasonably well with the currently available experimental data, although further refinements need to be made. The methodology illustrated in this article could be used for evaluation of the gene expression efficiency of other genetically engineered recombinants once such recombinants with certain gene-host cell systems are constructed.
    Additional Material: 9 Ill.
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  • 97
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 32 (1988), S. 716-718 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
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  • 98
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 32 (1988), S. 733-740 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Plasmid loss kinetics for Saccharomyces cerevisiae transformed with the 2-μm DNA-based-plasmid pUCKm8 were measured in nonselective and selective media. The plasmid pUCKm8 gives the organism two new phenotypes: resistance to the wide spectrum antibiotic G418 sulfate, and the ability to produce the enzyme, β-lactamase. Plasmid stability was determined using the production of β-lactamase as a marker. The effect of G418 on the growth rates of all organisms present in the culture and on plasmid stability was also determined. Mathematical models describing plasmid loss kinetics during exponential growth for both nonselective and selective conditions are used to simulate the experimental data. In nonselective medium, over 80% of the cells still exhibited the desired phenotype after 50 doublings. In medium containing G418, improvements in plasmid stability were only marginal due to the appearance of antibiotic-resistant cells.
    Additional Material: 17 Ill.
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  • 99
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 32 (1988), S. 760-763 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Cellulase producing activity of Trichoderma reesei QM9414 was examined under various agitation intensities and at the dissolved oxygen concentration above 3 ppm. The producing activity greatly depended upon the agitation intensity, and the dependence on the agitation was different for each cellulase-constituting component. The maximum producing activities of FPA, CM Case, and β-glucosidase were obtained under different agitation conditions, 1.0, 0.7, and 1.4 m/s in tip velocity, respectively. Intensive agitation brought about remarkable reduction in all cellulase components. The mycelial transformation through agitation intensity was also observed. Comparatively mild agitation of 0.3-1.0 m/s caused pellet formation as the culture progressed, although the pelletization was delayed with increasing agitation intensity. The behavior of the pelletization did not occur at 1.3 and 1.7 m/s throughout the course of cultivation, and under the latter agitation condition hyphae were broken up into short fragments. The cellulase producing activity is discussed in relation to such morphological changes.
    Additional Material: 5 Ill.
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  • 100
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 32 (1988), S. 786-796 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A population-balance model has been used to characterize continuous polyelectrolyte precipitation of egg white proteins. We have modeled the particle size distributions of aggregates formed under a range of mixing conditions. The models, accounting for aggregate growth (by both shear-driven and Brownian-like collisions), breakage (by hydrodynamic shear or aggregate-aggregate collisions), and birth (by the breakage of large aggregates), fit the data well. The kinetic constants show dependencies on shear rate and residence time that have not been previously theoretically predicted; these dependencies are due in part to aging effects on the aggregate. The model constants show a dominance of growth over breakage, supporting qualitative interpretations of the particle size distributions. A mechanism for growth-rate enhancement, caused by polymer extensions from the particle surfaces, produced improved model performance. A collisional breakage mechanism is supported.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
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