PMID:9074616

Citation	Bernardi, R, Rossi, L, Poirier, GG and Scovassi, AI (1997) Analysis of poly(ADP-ribose) glycohydrolase activity in nuclear extracts from mammalian cells. Biochim. Biophys. Acta 1338:60-8
Abstract	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 32P-labelled polymer as a 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 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, 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.
Links	PubMed
Keywords	Adenosine Diphosphate Ribose/analysis; Animals; Autoradiography; CHO Cells; Cell Nucleus/enzymology; Chromatography, Thin Layer; Cricetinae; Ethacridine/pharmacology; Etoposide/pharmacology; Glycoside Hydrolases/isolation & purification; Glycoside Hydrolases/metabolism; HL-60 Cells; HeLa Cells; Humans; Kinetics; NAD/metabolism; Neuroblastoma; Phosphorus Radioisotopes; Tumor Cells, Cultured
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
BOVIN:PARG	enables	GO:0004649: poly(ADP-ribose) glycohydrolase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete
BOVIN:PARG		GO:0004649: poly(ADP-ribose) glycohydrolase activity	ECO:0000314:		F		Figure 1 shows autoradiographic evaluation of PARG activity. The Poly(ADP-ribose) glycohydrolase (PARG) was purified from calf thymus	complete CACAO 8908
HUMAN:PARG		GO:0004649: poly(ADP-ribose) glycohydrolase activity	ECO:0000314:		F		Table 1	complete CACAO 8910
HUMAN:PARG		GO:0005634: nucleus	ECO:0000314:		C		Figure 1. Autoradiographic evaluation of PARG activity in total and nuclear extracts from HeLa cells	complete CACAO 9046
HUMAN:PARG	enables	GO:0004649: poly(ADP-ribose) glycohydrolase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete
HUMAN:PARG	part_of	GO:0005634: nucleus	ECO:0000314: direct assay evidence used in manual assertion		C		Seeded From UniProt	complete
CRIGR:G3H9W3	enables	GO:0004649: poly(ADP-ribose) glycohydrolase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete
CRIGR:G3H9W3	part_of	GO:0005634: nucleus	ECO:0000314: direct assay evidence used in manual assertion		C		Seeded From UniProt	complete
CRIGR:G3H9W3		GO:0004649: poly(ADP-ribose) glycohydrolase activity	ECO:0000314:		F		Table 1. From the autoradiographic results of PARG activity, hydrolysis ratio and specific activity were calculated in Chinese Hamster ovary cells.	complete CACAO 8911
CRIGR:G3H9W3		GO:0005634: nucleus	ECO:0000314:		C		Table 1. Glycohydrolase activity in the nucleus.	complete CACAO 9048

References

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