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  • Electronic Resource  (47)
  • Biochemistry and Biotechnology  (47)
  • 1
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 5 (1989), S. 224-232 
    ISSN: 0887-3585
    Keywords: effective pore's radius ; α-ketoglutarate dehydrogenase complex ; branched chain α-keto acid dehydrogenase complex ; electron microscopy ; multienzyme complex ; two-dimensional ; electrophoresis ; multienzyme complex ; aggregation of Pyruvate dehydrogenase complex ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: In the studies of the size and structure of multienzyme complexes, a procedure complementary to electron microscopy for determining the molecular dimensions of hydrated multisubunit complexes is needed. For some applications this procedure must be capable of detecting aggregation of complexes and must be applicable to impure preparations. In the present study, a procedure of two-dimensional agarose gel electrophoresis (2d-AGE) (Serwer, P. et al. Anal. Biochem. 152: 339-345, 1986) was modified and employed to provide accurate sizemeasurements of several classical multienzyme complexes. To improve band clarity and to achieve required gel pore sizes, a hydroxyethylated agarose was used. The effective pore's radius (PE) as a function of gel concentration was determined for this agarose inthe range of PE value needed for multienzyme complexes (effective radius, R = 10-30 nm). Appropriate conditions wereestablished to measure R value ± 1% of the pyruvate (PDC), α-ketoglutarate (α-KGDC), and the branched chain α-keto acid (BCDC) dehydrogenase multienzyme complexes; the accuracy of R was limited by the accuracy of the determinations of the R value for the sizestandards. The PDC from bovine heart was found to have an R = 22.4 ± 0.2 nm following cross-linking with glutaraldehyde that was necessary for stabilization of the complex. Dimers and trimers of PDC, present in the preparations used, were separated from monomeric PDCduring 2d-AGE. All R values for the enzyme complexes studied were agreement with, though more accurate than, R valuesobtained by use of electron microscopy. In contrast to this statement, the internal dihydrolipoyl transacetylase core of PDC (E2) had an R of 18.8 ± 0.2 nm using 2d-AGE, but 10.5 nm by electron microscopy. This observation confirms the proposal that the core of the PDC has externally projecting fibrous domains invisibleto electron microscopy.
    Additional Material: 7 Ill.
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  • 2
    ISSN: 0887-3585
    Keywords: β-adrenergic recepor ; chimeric proteins ; receptor subtypes ; ligand binding ; protein structure-function ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Pharmacological analysis of ligand binding to the β-adrenergic receptor (βAR) has revealed the existence of two distinct receptor subtypes (β1 and β2) which are the products of different genes. The predicted amino acid sequence of the β1 and β2 receptors differ by 48%. To identify the regions of the proteins responsible for determining receptor subtype, chimeras were constructed from domains of the human β1 and hamster β2 receptors. Analyses of the ligand-binding characteristics of these hybrid receptors revealed that residues in the middle portion of the βAR sequence, particularly around transmembrane regions 4 and 5, contribute to the subtype specific binding of agonists. Smaller molecular replacement of regions of the hamster β2AR with the analogous regions from the avian β1AR, however, failed to identify any single residue substitution capable of altering the subtype specificity of the receptor. These data indicate that, whereas sequences around transmembrane regions 4 and 5 may contribute to conformations which influence the ligand-binding properties of the receptor, the subtype-specific differences in amine-substituted agonist binding cannot be attributed to a single molecular interaction between the ligand and any amino acid residue which is divergent between the β1 and β2 receptors.
    Additional Material: 4 Ill.
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  • 3
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 41 (1993), S. 303-315 
    ISSN: 0006-3592
    Keywords: biological acid transformation ; sulfuric acid conversion ; sulfuric acid disposal ; sulfate-reducing bacteria ; dihydrogensulfide toxicity ; fixed-bed reactor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: As an alternative to the current disposal technologies for waste sulfuric acid, a new combination of recycling processes was developed. The strong acid (H2SO4) is biologically converted with the weak acid (CH3COOH) into two volatile weak acids (H2S, H2CO3) by sulfate-reducing bacteria. The transformation is possible without prior neutralization of the sulfuric acid. The microbially mediated transformation can be followed by physiochemical processes for the further conversion of the H2S.The reduction of sulfate to H2S is carried out under carbon-limited conditions at pH 7.5 to 8.5. A fixed-bed biofilm column reactor is used in conjunction with a separate gas-stripping column which was installed in the recycle stream. Sulfate, total sulfide, and the carbon substrate (in most cases acetate) were determined quantitatively. H2S and CO2 are continually removed by stripping with N2. Optimal removal is achieved under pH conditions which are adjusted to values below the pKa-values of the acids. The H2S concentration in the stripped gas was 2% to 8% (v/v) if H2SO4 and CH3COOH are fed to the recycle stream just before the stripping column.Microbiol conversion rates of 65 g of sulfate reduced per liter of bioreactor volume per day are achieved and bacterial conversion efficiencies for sulfate of more than 95% can be maintained if the concentration of undissociated H2S is kept below 40 to 50 mg/L. Porous glass spheres, lava beads, and polyurethane pellets are useful matrices for the attachment of the bacterial biomass. Theoretical aspects and the dependence of the overall conversion performance on selected process parameters are illustrated in the Appendix to this article. © 1993 John Wiley & Sons, Inc.
    Additional Material: 6 Ill.
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  • 4
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 50 (1996), S. 222-227 
    ISSN: 0006-3592
    Keywords: γ-poly(glutamate) ; γ-PGA ; Bacillus licheniformis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Bacillus licheniformis ATCC 9945A was grown on Medium E in batch fermentations in which the pH was maintained at 5.5., 6.5, 7.4, and 8.25. The effects of pH on cell growth, carbon source utilization, and γ-polyglutamic acid (γ-PGA) production, molecular weight, and polymer stereochemistry were determined. The γ-PGA yield was highest (15 g/L, 96 h growth time) at pH 6.5. The increase in γ-PGA formation at pH 6.5 corresponded with a relatively high specific production rate at high γ-PGA concentration (0.09 h-1, ∼15 g/L γ-PGA). In contrast, the specific γ-PGA production rates at fermentor pH values of 5.5 and 7.4 decreased significantly for γ-PGA fermentor yields 〉∼5 g/L. Interestingly, alteration of the medium pH had little to no significant effects on the product quality as measured by stereochemical composition and molecular weight. While glutamate and glycerol utilization were similar as a function of pH, citrate consumption increased at pH 6.5, indicating that the formation of γ-PGA from citrate at pH 6.5 was of increased importance. The effect of aeration was evaluated by increasing the agitation speed (250 to 800 rpm) and aeration rate (0.5 to 2.0 L/min) at pH 6.5, the pH of maximal γ-PGA production. Increased aeration resulted in doubling of the cell dry weights (2 to 4 g/L), increasing γ-PGA yields (6.3 to 23 g/L by 48 h) and increasing in the maximum γ-PGA-specific production rate (0.09 to 0.11 h-1). Other effects of increased agitation included a rapid depletion of glutamate and citrate (by 50 h) and a decrease in product molecular weight. Despite the increase in agitation and aeration, oxygen limitation of the culture was not avoided, because the partial pressure decreased to 〈1.0% by 29 h. © 1996 John Wiley & Sons, Inc.
    Additional Material: 2 Ill.
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  • 5
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 430-437 
    ISSN: 0006-3592
    Keywords: glucose ; glycerol ; γ-poly(glutamic acid) ; Bacillus licheniformis ATCC 9945a ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Bacillus licheniformis ATCC 9945a is one of the bacterial strains that produce γ-poly(glutamic acid) (γ-PGA). The use of carbohydrate medium components for γ-PGA production was explored. Cells were grown in shake flasks or in controlled pH fermentors using medium formulations that contain different carbon sources. During the cultivations, aliquots were removed to monitor cell growth, carbon utilization, polymer production, and polymer molecular weight. Glucose was a better carbon source than glycerol for cell growth. Furthermore, glucose was utilized at a faster rate than glycerol, citrate, or glutamate. However, by using mixtures of glucose and glycerol in medium formulations, the efficiency of γ-PGA production increased. For example, by increasing the glycerol in medium formulations from 0 to 40 g/L, the γ-PGA broth concentration after 96 h increased from 5.7 to 20.5 g/L. Considering that glycerol utilization was low for the glucose/glycerol mixtures studied, it was unclear as to the mechanism by which glycerol leads to enhanced product formation. Cell growth and concomitant γ-PGA production (12 g/L) at pH 6.5 was possible using glucose as a carbon source if trace amounts (0.5 g/L each) of citrate and glutamate were present in the medium. We suggested that citrate and glutamate were useful in preventing salt precipitation from the medium. In addition, glutamate may be preferred relative to ammonium chloride as a nitrogen source. The conversion of glucose to γ-PGA by the strain ATCC 9945a was believed to occur by glycolysis of glucose to acetyl-CoA and tricarboxylic acid (TCA) cycle intermediates that were then metabolized via the TCA cycle to form α-ketoglutarate, which is a direct glutamate precursor. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 430-437, 1998.
    Additional Material: 9 Ill.
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  • 6
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 9 (1996), S. 488-493 
    ISSN: 0952-3499
    Keywords: polyalanine β-sheet complexes ; structural characterization ; 5′-mononucleotide binding ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A study was initiated into the formation and stability of highly soluble β-sheet macrostructures. Such β-sheet macrostructures are useful model systems for the study of the biological function of the hydrophobic core of proteins and for the de novo design of novel catalytic mimics. In the current study, a 16-mer-alanine-based peptide (Ac-KA14K-NH2) that is highly water soluble and adopts an extremely stable macromolecular β-sheet structure was synthesized. A tyrosine-containing analog (Ac-KYA13K-NH2) was used to study the tertiary structure of the complex by circular dichroism spectroscopy, while the influence of the charges on the complex formation and binding affinity was evaluated using a zwitterionic analog (Ac-KEA13KE-NH2). Both the secondary and tertiary structures of the β-sheet complex were stable to denaturants, as demonstrated by far- and near-ultraviolet circular dichroism spectroscopy. Binding studies with mononucleotides have shown that the β-sheet complex binds to molecules through both hydrophobic and electrostatic interactions. These intrinsic properties were found to be a prerequisite for the observed enhanced cleavage of phosphodiester bonds.
    Additional Material: 5 Ill.
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  • 7
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 5 (1992), S. 1-8 
    ISSN: 0952-3499
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Glutaraldehyde-polymerized human splenic galaptin, a β-galactoside-binding lectin, was demonstrated to have enhanced hemagglutinating and asialofetuin binding activity relative of native dimeric galaptin when these lectins were present in solution. The polymerized lectin consisted primarily of 2-, 4- and 12-membered species after reductive alkylation. Both forms of galaptin bound, at 4 °C, to saturable B lymphoblastoid cell surface receptors. Estimates obtained by Scatchard analyses, with the binding data expressed in terms of 14.5 kDa subunit molarity, were 5 × 107 binding sites/cell with affinity constant Ka = 2.2 × 105 M for dimeric galaptin and 17 × 107 binding sites/cell with Ka = 3.4 × 105 M-1 for polymeric galaptin. Both forms of galaptin adsorbed to polystyrene with high efficiency; however, only plastic-adsorbed polymeric galaptin mediated adhesion of lymphoblastoid cells. Cell adhesion was inhibited by lactose. Plastic-adsorbed polymeric galaptin bound asialofetuin more efficiently than dimeric galaptin. Asialofetuin binding was inhibited 65% and 30-50% by lactose for plastic-adsorbed polymeric and dimeric galaptin, respectively, Native fetuin bound to the adsorbed dimeric galaptin in a lactose-insensitive manner. These data indicate that cell surface receptor-galaptin interaction is carbohydrate specific whereas polystyrene-adsorbed galaptin may demonstrate protein-protein interactions with soluble ligands.
    Additional Material: 6 Ill.
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  • 8
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 7 (1986), S. 487-491 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Computer simulation is used to analyze a system of two electrophoretic columns coupled by mixing the anolyte of one with the catholyte of the other. A mathematical model is presented which is used to predict the pH gradients formed by monovalent buffers in this system, when the currents in the columns are unequal. In the column with the higher current a pH gradient is created which increases from anode to cathode and is potentially useful for isoelectric focusing. The breadth of this gradient is dependent upon the ratio of the currents. The function of the second column is the compensation of buffer migration which occurs in the first column, thereby maintaining constant electrolyte composition. The effects of buffer pKs and mobilities are evaluated.
    Additional Material: 5 Ill.
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  • 9
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 11 (1990), S. 292-298 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The isotachophoretic behavior of serum albumin is examined for three anionic and one cationic electrolyte systems by (i) computer simulation, (ii) capillary isotachophoresis (ITP) and (iii) continuous flow ITP. The theoretical relationship between pH of the leading electrolyte and the steady state protein plateau concentration is presented for one of the anionic systems. With leading ion concentrations of the order of 10 mm, experimental protein plateau concentrations of 1.3-2.3 % w/v are obtained. The computer predictions are approximately half these values.
    Additional Material: 7 Ill.
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  • 10
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A commercially available electrooptical device, coupled to a microcomputer, is capable of analyzing densitometric values with a resolution of 280 × 128 points (35 840 individual points). A complex autoradiogram of a two-dimensional electrophoretic gel is completely sorted into X and Y coordinates and exposure values differeing by as little as 0.01 O.D. (optical density) units or over an O.D. range of 0 to 4.00 in less than 2 min. For other purposes such as comparison of two gels, immunoelectrophoretic patterns, enzyme linked immunosorbent assay or microtitration plates, dot-blot assays, or tissue samples, a comparison may be made and all differences stored in the computer in less than one second and further decisions or computer-driven actions decided on that basis.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
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