Isolation and purification of poly(ADP-ribose) glycohydrolase from pig thymus (original) (raw)
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Analysis of poly(ADP-ribose) glycohydrolase activity in nuclear extracts from mammalian cells
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1997
. We have analysed poly ADP-ribose glycohydrolase, the enzyme responsible for in vivo degradation of ADP-ribose polymers, by means of a biochemical assay based on the capacity of the enzyme to use a synthetic 32 P-labelled polymer as a Ž . substrate. The visualization of the reaction has been achieved by separation of poly and mono ADP-ribose by thin-layer chromatography followed by autoradiography, whereas polymer hydrolysis has been quantified by counting the spots Ž . corresponding to poly and mono ADP-ribose . By addition of the enzyme inhibitor ethacridine to the reaction mixture, we have confirmed the specificity of the procedure we have developed. The protocol has been applied to study the specific activity of glycohydrolase in nuclear extracts from different mammalian cell lines and to an apoptotic experimental system, namely HL60 cells treated with etoposide. We have observed the activation of the enzyme after a two-hour drug treatment, Ž . that is concomitant with the activation of poly ADP-ribose polymerase, the enzyme which synthesizes the polymer. These data suggest a precise regulation of ADP-ribosylation process during cell death by apoptosis.
A new method for the assay of poly(adenosine diphosphate ribose) glycohydrolase activity
Analytical Biochemistry, 1975
Rib was estimated by measuring the radioactivity in aliquots of formic acid extract. Oligomers or polymers of ADP-Rib can be utilized as substrates since the reaction rates were the same with either compound. A method to determine phosphodiesterase and glycohydrolase activities was established. These two enzymic activities were distinguished by treating the products of the reactions with alkaline phosphatase and by differential extraction of the adsorbed reaction products on Dowex with 0.5 M and 6 M formic acid. Eukaryotic cells contain a nuclear enzyme that catalyzes the conversion of NAD into poly(ADP-Rib)' with the concomitant elimination of nicotinamide (I-5). The repeating units of ADP-Rib are linked together by a glycosidic bond of ribosyl-1,2-ribose (3,5). The characteristics of the polymer and properties of the polymerizing enzyme have been reported (6-9). This biopolymer is hydrolyzed by two types of enzymes, namely, phosphodiesterase and glycohydrolase. The pyrophosphate bond is hydrolyzed by venom and rat-liver nuclear phosphodiesterase (2,4,10,11). The products of the reaction are AMP, PR-AMP and ribose-5'-phosphate. The chain length of the polymer can be calculated from the relative amounts of PR-AMP and AMP (12). Recently, a new enzymatic reaction has been reported which splits the ribose-ribose linkage between two ADP-Rib units with the release of only ADP-Rib
Molecular Biology Reports, 1988
Poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase activities were both investigated in chicken erythroblasts transformed by Avian Erythroblastosis Virus. Respectively 21% and 58% of these activities were found to be present in the post-mitochondrial supernatant (PMS). Fractionation of the PMS on sucrose gradients and poly(A § mRNA detection by hybridization to [3H] poly(U) show that cytoplasmic poly(ADPribose) polymerase is exclusively localized in free mRNP. The glycohydrolase activity sedimented mostly in the 6 S region but 1/3 of the activity was in the free mRNP zone. Seven poly(ADP-ribose) protein acceptors were identified in the PMS in the Mr 21000-120000 range. The Mr 120000 protein corresponds to automodifled poly(ADP-ribose) polymerase. A Mr 21000 protein acceptor is abundant in PMS and a Mr 34000 is exclusively associated with ribosomes and ribosomal subunits. The existence of both poly(ADP-ribose) polymerase and glycohydrolase activities in free mRNP argues in favour of a role of poly(ADP-ribosylation) in mRNP metabolism. A possible involvement of this post translational modification in the mechanisms of repressionderepression of mRNA is discussed.
Poly(ADP-ribose) Glycohydrolase Is Present and Active in Mammalian Cells as a 110-kDa Protein
Experimental Cell Research, 1999
Poly(ADP-ribose) glycohydrolase (PARG) is the major enzyme responsible for the catabolism of poly-(ADP-ribose), a reversible covalent-modifier of chromosomal proteins. Purification of PARG from many tissues revealed heterogeneity in activity and structure of this enzyme. To investigate PARG structure and localization, we developed a highly sensitive onedimensional zymogram allowing us to analyze PARG activity in crude extracts of Cos-7, Jurkat, HL-60, and Molt-3 cells. In all extracts, a single PARG activity band corresponding to a protein of about 110 kDa was detected. This 110-kDa PARG activity was found mainly in cytoplasmic rather than in nuclear extracts of Cos-7 cells.
Mutation Research Letters, 1990
Poly(ADP-ribosyl)ation of nuclear proteins is catalyzed by poly(ADP-ribose) polymerase. This enzyme is involved in the regulation of basic cellular functions of DNA metabolism. DNA breaks induced by DNAdamaging agents trigger the activation of poly(ADP-ribose) polymerase increasing its endogenous level. This increase modifies the pattern of poly(ADP-ribosyl)ated chromatin proteins. In this paper we describe a procedure for the isolation of intact nuclei from rat liver to be used for the endogenous activity assay. Artifactual activation of the enzyme was avoided since a very low level of DNA-strand breaks occurs during the isolation of nuclei. We present a series of experiments which prove the ability of this procedure to detect increases in endogenous liver activity without modification of the total level. The application of this technique can be useful for a better understanding of the role of early changes in poly(ADP-ribose) polymerase level in physiological conditions and during exposure to DNA-damaging agents.
Purification and Properties of Poly(ADP-ribose) Polymerase from Pig-Thymus Nuclei
European Journal of Biochemistry, 1978
The nuclear enzyme poly(ADP-ribose) polymerase has been purified about 9200-fold from pig thymus nuclei with a 46% yield. An aqueous organic solvent system was used for the isolation of the polymerase from nuclei and for its purification by chromatography at sub-zero temperatures. Electrophoretic analysis under both denaturing and non-denaturing conditions revealed a single protein band suggesting that the preparation was homogeneous and that the enzyme is composed of one polypeptide chain. The molecular weight estimated from sodium dodecyl sulphate-/polyacrylamide gel electrophoresis was 63 500 and from gel filtration through columns of Sephadex G-100, 58 000. The enzyme preparation was free from poly(ADP-ribose)-degrading enzymes and from DNA. The purified polymerase showed an absolute requirement for both DNA and histones. The maximal specific activity of the homogeneous preparation measured by the standardized assay, was 20.7 pmol NAD* incorporated x min-' x mg-' of protein at 37 "C. Amino-terminal group analysis with dansyl chloride did not reveal a terminal amino acid suggesting that the amino-terminal group may be blocked. In the presence of histones, the K,,, for NAD' was 23 pM.
Preferential Perinuclear Localization of Poly(ADP-ribose) Glycohydrolase
Experimental Cell Research, 1999
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.
European Journal of Biochemistry, 1976
Poly(ADP-ribose) polymerase with a high specific activity was obtained from Ehrlich ascites tumor cells by extraction of nuclei with 175 mM potassium phosphate, followed by chromatography on DNA-agarose. Electrophoretic analysis indicated that the preparation contained two proteins, one of which was shown to catalyze the synthesis of poly(ADP-ribose). As expected from results obtained by other workers, the synthesis was inhibited by nicotinamide and thymidine, and stimulated by DNA. Addition of histones gave inhibition of the synthesis, unless DNA was present in the reaction mixture.