Radioiodination of microgram quantities of ribosomal proteins from polyacrylamide gels
Abstract
A method has been developed for radiolabeling small amounts of ribosomal proteins extracted from polyacrylamide gels with potassium [125]Iiodide. The procedure was used to label even those proteins which lack tyrosine and histidine residues by the modification of proteins with methyl p-hydroxybenzimidate. Specific radioactivities obtained range from 20,000 to 200,000 cpm/μg. The method has been used in the identification of eukaryotic ribosomal proteins from rabbit reticulocytes separated by polyacrylamide/sodium dodecyl sulfate gel electrophoresis.
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Proteins from the organic matrix of core-top and fossil planktonic foraminifera
1990, Geochimica et Cosmochimica ActaOrganic constituents isolated from the tests (shells) of six species of core-top planktonic foraminifera, ranging in age between 2 and 4 Ka BP, consist of a heterogeneous mixture of proteins and polypeptides. At least seven discrete polypeptides are present as indicated by reverse phase HPLC and by gel electrophoresis. High percentages of aspartic acid and glutamic acid characterize one class of protein, while glycine, serine, and alanine-rich proteins dominate in a second class. Similar HPLC Chromatographie elution profiles are observed for all species analyzed, varying only in intensity of the peaks and in amino acid composition from species to species. The approximate molecular weights of two major fossil proteins ranged between 50,000 and 70,000 daltons. A comparison of 2–4 and 300 Ka Bp samples shows that while most of the polypeptides are present in both samples, some acidic polypeptides are not present in the older sample. These data suggest that some of the acidic polypeptides may be more soluble than other fractions and are lost more quickly from the test. The remaining hydrophobic, possibly more insoluble, polypeptides may be preserved in much older specimens and may be useful in tracing phylogeny of the planktonic foraminifera.
Amino acid analyses of total test extracts before and after dialysis demonstrate that some acidic amino acids, particularly aspartic acid, and possibly peptides less than 6000–8000 daltons are lost during dialysis. Although a large percentage of these components are undoubtedly from the original organic matrix, at this point adsorbed components cannot be ruled out. These data caution against the use of total amino acid compositions in biogeochemical studies.
Identification of proteins crosslinked to RNA in 40S ribosomal subunits of Saccharomyces cerevisiae
1989, BiochimieRNA-protein crosslinks were introduced into the 40S ribosomal subunits from Saccharomyces cerevisiae by mild UV treatment. Proteins crosslinked to the 18S rRNA molecule were separated from free proteins by repeated extraction of the treated subunits and centrifugation in glycerol gradients. After digestion with RNase to remove the RNA molecules, proteins were radio-labeled with 125I and identified by electrophoresis on two-dimensional polyacrylamide gels with carrier total 40S ribosomal proteins and autoradiography. Proteins S2, S7, S13, S14, S17/22/27, and S18 were linked to the 18S rRNA. A shorter period of irradiation resulted in crosslinking of S2 and S17/22/27 only. Several of these proteins were previously demonstrated to be present in ribosomal core particles or early assembled proteins.
Labeling of monoclonal antibodies with radionuclides
1989, Seminars in Nuclear MedicineAntibodies, specifically monoclonal antibodies, are potentially very useful and powerful carriers of therapeutic agents to target tissues and diagnostic agents. The loading or charging of antibodies with agents, especially radiotracers, is reviewed here. The choice of radiosotope for immunodetection and/or immunotherapy is based on its availability, half-life, nature of the radiation emitted, and the metabolic pathways of the radionuclide in the body. Most important of all are the derivatization techniques available for labeling the antibody with the given radionuclide. Isotopes of iodine and divalent metal ions are the most, commonly used radionuclides. Antibodies labeled with iodine at tyrosine residues are metabolized rapidly in vivo. This leads to the incorporation of metabolized radioactive iodine into various tissues, mainly the thyroid gland and stomach, and to the accumulation of high levels of circulating iodine in the blood, which masks tumor uptake considerably. To overcome these limitations, the use of iodohippurate as an iodine-anchoring molecule, to the protein should be considered. When divalent or multivalent metal ions are used as the preferred, radionuclide, bifunctional chelating reagents such as ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA) are first coupled to the protein or antibody. These chelating molecules are attached to the protein by formation of an isopeptide linkage between the carboxylate of the chelating reagent and the amino group of the protein. Several procedures are available to generate the isopeptide linkage. When the anchoring of the chelating agent through isopeptide linkage results in the inactivation of the antibody, periodate oxidation of the carbohydrate moiety of the antibody, followed by reductive coupling of chelator, could be considered as an alternative. There is still a need for better, simpler, and more direct methods for labeling antibodies with radionuclides.
Structure of monkey kidney cell RNA polymerase II: Characterization of RNA polymerase associated with SV40 late transcriptional complexes
1986, Archives of Biochemistry and BiophysicsThree subspecies of RNA polymerase II have been described in eucaryotic cells and designated IIO, IIA, and IIB. Although their relative proportions vary among different sources, RNA polymerases IIA and IIB constitute the bulk of most purified RNA polymerase II preparations. Antibodies against calf thymus RNA polymerase II were used to estimate the amount of polymerase II subspecies in monkey kidney cells, isolated nuclei, and SV40 late transcriptional complexes. We have found that RNA polymerase IIO is present in whole cells and isolated nuclei in higher proportions than previously reported. Subspecies IIO was found associated with SV40 minichromosomes engaged in transcription during late lytic infection. The observation that RNA polymerase IIO is associated with the cellular chromatin and SV40 minichromosomes suggest that this form of the enzyme is the subspecies active in in vivo transcription.
Selective high-efficiency cross-linking of mammalian ribosomal proteins with cleavable thiol-directed heterobifunctional reagents: Identification and binding directions of major protein complexes
1986, Biochimica et Biophysica Acta (BBA)/Protein Structure and MolecularRat liver and mouse ascitic tumour ribosomal proteins are cross-linked selectively in good yield with the newly developed cleavable heterobifunctional reagents 2-(4-hydroxy-2-maleimidophenylazo)benzoic acid N-hydroxysuccinimide ester (reagent A) and 4-(4-hydroxy-3-maleimidophenylazo)[carboxyl-14C]benzoic acid N-hydroxysuccinimide ester (reagent B). The primary function of the reagents, an N-arylated maleimide, binds quantitatively at low pH to accessible cysteine groups. After eliminating the free reagent, the pH is increased to make the secondary function, a juxtanuclear aroyl ester, reactive against neighboring amino groups, essentially lysine. The spacer, 4-phenylazophenol, is readily cleaved by reduction with dithionite. The ranges of cross-linking of the two reagents are approx. 8 and 12 Å, respectively. Using the radiolabelled reagent B the secondarily attached protein (and its contact sequence) is made recognizable even in trace amounts. The order of binding of the interacting proteins is thereby established. The two reagents produce similar, but not identical, patterns of selective cross-linking. The following protein complexes are readily observed after conventional staining. With reagent A: S8-S11, L4-L14, L4-L18, L6-L29 and L21-L18a. With the radioactive, longer-range reagent B: L4 ← L13a, L4 → L18, L4 ← L18a, L4 ← L26, L6 ← L29, L14 ← L13a, L21 ← L18a and L27 ← L30 (arrows indicating the direction of binding). Ternary and quaternary complexes are also obtained, especially of the large protein L4. With both reagents a protein designated L6′ is cross-linked to L23. The predominant cross-linked complexes can be obtained on a preparative scale for isolation and characterization of contact sequences by optional fragmentation and fractionation methods.
Isolation and characterization of two cyanogenic β-glucosidases from flax seeds
1985, Archives of Biochemistry and BiophysicsTwo cyanogenic β-glucosidases, linustatinase and linamarase, were isolated and purified from flax seeds (Linum ussitatissimum). They catalyze the sequential hydrolysis of linustatin and neolinustatin to yield acetone and methylethyl ketone cyanohydrins, respectively. The purification procedure for linustatinase involved acetone extraction, precipitation by polyethyleneimine and ammonium sulfate (40–80% saturation), and Red A gel, concanavalin A-Sepharose, and PBE 94 column chromatography; that for linamarase was similar except that polyethyleneimine precipitation was eliminated and DE-52 and Sepharose CL-6B replaced Red A gel column chromatography. The native substrates neolinustatin and linamarin were used for the assay during purification. Both proteins were purified to electrophoretic homogeneity. Linustatinase is an αβ dimer (molecular weights of α and β = 39,000 and 19,000, respectively) while linamarase appears to be an α5β5 decamer (molecular weights of α and β = 62,500 and 65,000, respectively). Both enzymes contain mannose or glucose. Linustatinase exists in five different isozymic forms (isoelectric points between 7 and 8) whereas linamarase occurs in one major form (isoelectric point 4 to 5). The kinetic parameters of the two enzymes are similar: acidic pH optima, Km's in the millimolar range, and competitive inhibition by δ-gluconolactone, a transition state analog. The presence of an aglycone structure in the substrates is important for both enzyme activities. In addition, both enzymes are specific towards the β-glycosidic linkage; linustatinase (a β-bis-glucosidase) readily hydrolyzes β-bisglucosides with 1,6 and 1,3 linkages whereas linamarase (a β-monoglucosidase) exhibits little activity towards these substrates.