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Query: EC:3.2.1.143 (poly(ADP-ribose) glycohydrolase)
 208 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Rat peritoneal macrophages are known to contain a chymotrypsin-like neutral protease associated with a specific inhibitor. By homogenizing the cells in 0.25 M sucrose (pH 8.0) containing 0.5% Triton X-100, both the protease and the inhibitor were found to be localized in the nuclei, particularly in chromatin. The inhibitory factor in chromatin was then separated from the protease by hydroxylapatite gel chromatography in the presence of 2 M NaCl and 5 M urea. The inhibitor fraction obtained was deproteinized by digestion with Pronase and subsequent extraction with phenol; these treatments did not alter the inhibitory potency. The deproteinized inhibitor fraction had a UV absorption ratio, A280/A260, of 0.61, but it was resistant to digestion with various nucleases, including DNase 1, nuclease P1, and snake venom phosphodiesterase. However, when it was incubated with poly(ADP-ribose) glycohydrolase from calf thymus, the inhibitory potency was markedly decreased. An authentic poly(ADP-ribose), with a mean chain length of approximately 30 ADP-ribose units, produced significant inhibition of the neutral protease isolated from macrophage chromatin. No such inhibition was produced by DNA, single-stranded DNA, RNA, polyadenylate, polyuridylate, polycytidylate, or monomeric ADP-ribose.
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PMID:A chromatin-bound neutral protease and its inhibitor in rat peritoneal macrophages. 71 9

The preparation of quantities of poly(ADP-ribose) glycohydrolase sufficient for detailed structural and enzymatic characterizations has been difficult due to the very low tissue content of the enzyme and its lability in late stages of purification. To date, the only purification of this enzyme to apparent homogeneity has involved a procedure requiring 6 column chromatographic steps. Described here is the preparation of an affinity matrix which consists of ADP-ribose polymers bound to dihydroxyboronyl sepharose. An application is described for the purification of poly(ADP-ribose) glycohydrolase from calf thymus in which a single rapid affinity step was used to replace 3 column chromatographic steps yielding enzyme of greater than 90% purity with a 3 fold increase in yield. This matrix should also prove useful for other studies of ADP-ribose polymer metabolism and related clinical conditions.
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PMID:An affinity matrix for the purification of poly(ADP-ribose) glycohydrolase. 239 36

It has been demonstrated recently by Poirier et al. (Poirier, G. G., de Murcia, G., Jongstra-Bilen, J., Niedergang, C., and Mandel, P. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3423-3427) that poly(ADP-ribosyl)ation of pancreatic nucleosomes causes relaxation of the chromatin superstructure through H1 modification. The in vitro effect of poly(ADP-ribose) synthesis and degradation on calf thymus chromatin was investigated by the time course incorporation of ADP-ribose, electron microscopy, analytical ultracentrifugation, and autoradiography of the protein acceptors. Purified calf thymus poly(ADP-ribose) polymerase and partially purified bull testis poly(ADP-ribose) glycohydrolase were used. Degradation of ADP-ribose units on hyper(ADP-ribosyl)ated H1 by poly(ADP-ribose) glycohydrolase restores the native condensed chromatin superstructure. This reversible conformational change induced by poly(ADP-ribosyl)ation on nucleosomal arrangement could be one of the mechanisms by which the accessibility of DNA polymerases and/or excision-repair enzymes is favored, the native structure being fully restorable.
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PMID:Modulation of chromatin superstructure induced by poly(ADP-ribose) synthesis and degradation. 308 93

Poly(ADP-ribose) synthetic activity in isolated nucleoli from rapidly growing mouse ascites tumor cells and ADP-ribosylation of the nucleolar proteins in vitro were studied. The specific activity of the synthesis in the nucleoli was significantly higher than that in the chromatin. The optimum magnesium and NAD+ concentrations, and the effect of RNase treatment on the reaction in the nucleoli were also distinctly different from those in the chromatin. Hydrolysis of the reaction product of the nucleoli with snake venom phosphodiesterase and with calf thymus poly(ADP-ribose) glycohydrolase yielded 5'-AMP and 2'-(5"-phosphoribosyl))5'-AMP, and ADP-ribose, respectively. The average chain length of the polymer formed in the nucleoli was found to be about 4 as a whole, but the distribution was heterogenous, from 1.2 to over 12. Analysis of ADP-ribosylated proteins in the nucleoli by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that several non-histone proteins with molecular weights of over 100,000 were highly ADP-ribosylated compared with other proteins including histones. This pattern was also different from that of the chromatin. These experimental results demonstrate that the nucleoli are independent from the chromatin as regards poly(ADP-ribose) synthesis in vitro.
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PMID:Poly(ADP-ribose) synthesis in nucleoli and ADP-ribosylation of nucleolar proteins in mouse ascites tumor cells in vitro. 728 63

Poly(ADP-ribosyl)ation metabolism, a post-translational modification, involves two nuclear enzymes. Poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG) are responsible for the anabolism and catabolism of poly(ADP-ribose) polymer, respectively. PARG, despite being less abundant than PARP, is a crucial determinant of polymer metabolism which is known to be implicated in DNA repair and other cellular processes. Here, we describe modifications to improve the purification of PARG from calf thymus, in terms of both quantity and quality, which would allow biochemical and immunological studies. We also developed a zymogram to identify functional polypeptides exhibiting PARG activity. Purified and crude enzyme preparations from calf thymus were electrophoresed in two-dimensional gels. Samples were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis containing the polymer substrate in the form of automodified PARP after a nonequilibrium pH gradient electrophoresis. After renaturation of PARG in the gel, four isoforms of activity were clearly detected in the purified enzyme preparation. Even in the crude extract of the tissue, we could observe the major isoform of PARG. This technique will permit a better understanding of poly(ADP-ribose) catabolism and better characterization of PARG isoforms.
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PMID:Purification of poly(ADP-ribose) glycohydrolase and detection of its isoforms by a zymogram following one- or two-dimensional electrophoresis. 807 79

A poly(ADP-ribose) glycohydrolase was purified more than 5,000-fold to apparent homogeneity from pig testis nuclei with a yield of 16%. A protein band, whose molecular mass (Mr) was estimated to be 58,000, detected by SDS-polyacrylamide gel electrophoresis of the purified preparation, was shown to have glycohydrolase activity upon assay by the renaturation method. A native Mr of 51,000 was determined by gel permeation. This polypeptide is a basic protein with a pI value of 8.8. The mode of hydrolysis of poly(ADP-ribose) [(ADP-ribose)n] by this enzyme is exoglycosidic, yielding ADP-ribose as the final product. The Km value for (ADP-ribose)n (average chain length, n = 15) is 5.4 microM and the Vmax of its hydrolysis is 34.5 micromol x min(-1) x mg protein(-1). The optimum pH for enzyme activity is 7.2. Low concentrations (50 approximately 150 mM) of monovalent salts stimulate the enzyme activity. The poly(ADP-ribose) glycohydrolase present in pig testis nuclei has some properties different from either nuclear poly(ADP-ribose) glycohydrolase (type I) or cytoplasmic poly(ADP-ribose) glycohydrolase (type II), purified previously from several tissues including pig thymus, guinea pig liver, calf thymus, human erythrocytes, and placenta. These differences suggest the tissue specificity of poly(ADP-ribose) glycohydrolase.
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PMID:Properties of poly(ADP-ribose) glycohydrolase purified from pig testis nuclei. 895 Oct 44

The synthesis and rapid turnover of ADP-ribose polymers is an immediate cellular response to DNA damage. We report here the isolation and characterization of cDNA encoding poly(ADP-ribose) glycohydrolase (PARG), the enzyme responsible for polymer turnover. PARG was isolated from bovine thymus, yielding a protein of approximately 59 kDa. Based on the sequence of oligopeptides derived from the enzyme, polymerase chain reaction products and partial cDNA clones were isolated and used to construct a putative full-length cDNA. The cDNA of approximately 4.1 kilobase pairs predicted expression of a protein of approximately 111 kDa, nearly twice the size of the isolated protein. A single transcript of approximately 4. 3 kilobase pairs was detected in bovine kidney poly(A)+ RNA, consistent with expression of a protein of 111 kDa. Expression of the cDNA in Escherichia coli resulted in an enzymatically active protein of 111 kDa and an active fragment of 59 kDa. Analysis of restriction endonuclease fragments from bovine DNA by Southern hybridization indicated that PARG is encoded by a single copy gene. Taken together, the results indicate that previous reports of multiple PARGs can be explained by proteolysis of an 111-kDa enzyme. The deduced amino acid sequence of the bovine PARG shares little or no homology with other known proteins. However, it contains a putative bipartite nuclear location signal as would be predicted for a nuclear protein. The availability of cDNA clones for PARG should facilitate structure-function studies of the enzyme and its involvement in cellular responses to genomic damage.
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PMID:Isolation and characterization of the cDNA encoding bovine poly(ADP-ribose) glycohydrolase. 911 50

The transient nature of poly(ADP-ribosyl)ation, a posttranslational modification of nuclear proteins, is achieved by the enzyme poly(ADP-ribose) glycohydrolase (PARG) which hydrolyzes the poly(ADP-ribose) polymer into free ADP-ribose residues. To investigate the molecular size and localization of PARG, we developed a specific polyclonal antibody directed against the bovine PARG carboxy-terminal region. We found that PARG purified from bovine thymus was recognized as a 59-kDa protein, while Western blot analysis of total cell extracts revealed the presence of a unique 110-kDa protein. This 110-kDa PARG was mostly found in postnuclear extracts, whereas it was barely detectable in the nuclear fractions of COS7 cells. Further analysis by immunofluorescence revealed a cytoplasmic perinuclear distribution of PARG in COS7 cells overexpressing the bovine PARG cDNA. These results provide direct evidence that PARG is primarily a cytoplasmic enzyme and suggest that a very low amount of intranuclear PARG is required for poly(ADP-ribose) turnover.
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PMID:Preferential perinuclear localization of poly(ADP-ribose) glycohydrolase. 1047 22