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Query: EC:2.7.7.8 (polynucleotide phosphorylase)
 723 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This report describes structural studies on purified polynucleotide phosphorylase from C. perfringens. A method is described for the purification of the enzyme which yields a product equivalent in activity to the native polynucleotide phosphorylase from E. coli. These studies revealed a molecular heterogeneity arising from successive stages of proteolysis, to which this enzyme is especially sensitive; unusally, the enzyme is obtained as a mixture of variable proportions of the native and proteolysed forms. We found in all cases a trimeric basic structure composed of the native (alpha) or proteolysed (lapha) or proteolysed (alpha', alpha") catalytic sub-units, However, the enzyme is rather easily dissociated into its sub-units, a phenomenon which seems to accompany proteolysis (Table). Under the action of either endogenous proteases or trypsin, two enzymatic forms are obtained: their quaternary structures seem analogous, but they differ in their catalytic properties from each other and from the initial enzyme. With some care at each step of purification, the polynucleotide phosphorylase of E. coli can be obtained exclusively in its native form. The greater susceptibility to proteolysis of the enzyme from C. perfrigens and the relationship between such degradation and quaternary structure seem to be at the origin of the peculiar behavior of this polynucleotide phosphorylase.
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PMID:[Quaternary structure and proteolysis of the polynucleotide phosphorylase from C. perfringens]. 21 36

1. A new method has been developed for the preparation in good yield of highly purified Azotobacter vinelandii polynucleotide phosphorylase in its reduced form. 2. Aging or digestion with trypsin causes the enzyme to develop a primer requirement that is not eliminated by beta-mercaptoethanol. 3. The development of a primer requirement is accompanied by marked changes of the electrophoretic mobility of the enzyme in polyacrylamide gels. 4. The enzyme is inactivated by aerial oxidation or thiol-specific reagents. The lost activity is restored by beta-mercaptoethanol, but not by oligonucleotide primers.
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PMID:Preparatin, proteolysis and reversible oxidationof highly purified Azotobacter vinelandii polynucleotide phosphorylase. 549 50

1. Trypsin digestion of Micrococcus lysodeikticus polynucleotide phosphorylase (nucleoside diphosphate-polynucleotide nucleotidyltransferase) causes a progressive increase in electrophoretic mobility in polyacrylamide gels of the single active degradation product. 2. A marked increase in primer requirement for CDP polymerization occurs before a more mobile product is formed. 3. alpha-Chymotrypsin digestion yields a product that separates into several active species on polyacrylamide-gel electrophoretograms. 4. No separation of ADP-and CDP-polymerization activities occurs during electrophoresis after either trypsin or alpha-chymotrypsin treatment.
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PMID:A study by polyacrylamide-gel electrophoresis of the effect of proteolysis on Micrococcus lysodeikticus polynucleotide phosphorylase. 570 78

1. Treatment of Micrococcus lysodeikticus polynucleotide phosphorylase (nucleoside diphosphate-polynucleotide nucleotidyltransferase) with trypsin causes a preferential loss of its cytidine diphosphate and uridine diphosphate polymerization activities. 2. The phosphorolytic activity of the enzyme towards polycytidylic acid is unaffected in conditions in which the cytidine diphosphate-polymerization activity without added primer is virtually abolished. 3. The treated enzyme retains its altered pattern of activities when purified fivefold by gel filtration. 4. The effect on the cytidine diphosphate-polymerization activity is due, in part, to a large increase in primer requirement as a result of proteolysis, and is qualitatively independent of the state of purity of the polynucleotide phosphorylase. 5. The enzyme is protected from trypsin degradation by nucleic acids, polynucleotides and nucleoside disphosphates. 6. A similar, but less marked differential effect, is caused by alpha-chymotrypsin.
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PMID:The effect of trypsin digestion on the activities of polynucleotide phosphorylase. 605 26

Tropheryma whipplei, the agent of Whipple's disease, is a gram-positive rod-shaped bacterium that belongs to the group of actinobacteria. In order to produce monoclonal antibodies (MAbs) against this bacterium, we inoculated mice with two different strains, Slow2 and Endo5. We produced 13 and 10 MAbs against Slow2 and Endo5, respectively. Nine of the Slow2 MAbs and seven of the Endo5 MAbs recognized a 58-kDa epitope. In addition, three other Endo5 MAbs detected a unique 84-kDa epitope. These MAbs were species specific, as they did not react with a selection of 22 different bacterial species, but they were not strain specific, as they did react with six other strains of T. whipplei. Two-dimensional gel electrophoresis (2-DE) was combined with mass spectrometry (MS) to identify the 58-kDa and 84-kDa epitopes recognized by MAbs. After trypsin in-gel digestion of the spot, the 58-kDa protein was identified as an ATP synthase F1 complex beta chain, whereas the 84-kDa protein was identified as a polyribonucleotide nucleotidyltransferase by MS with matrix-assisted laser desorption ionization-time of flight. In an in vitro model, one of these MAbs allowed good detection of T. whipplei in stool samples, contrary to a rabbit polyclonal antibody, which led to high fluorescent background. In the prospective studies, the produced MAb will be tested for detection of T. whipplei in clinical samples, and the gene coding for identified 58-kDa and 84-kDa antigens will be tentatively cloned and then tested for its use in a diagnostic enzyme-linked immunosorbent assay for Whipple's disease.
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PMID:Production of monoclonal antibodies to Tropheryma whipplei and identification of recognized epitopes by two-dimensional electrophoresis and mass spectrometry. 1698 20