Analysis of poly(ADP-ribose) glycohydrolase activity in nuclear extracts from mammalian cells.
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.
Bernardi
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R
Istituto di Genetica Biochimica ed Evoluzionistica del CNR, Pavia, Italy.
Rossi
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L
Poirier
G G
GG
Scovassi
A I
AI
D000246:Adenosine Diphosphate Ribose / Q000032:analysis; D000818:Animals; D001345:Autoradiography; D016466:CHO Cells; D002467:Cell Nucleus / Q000201:enzymology*; D002855:Chromatography, Thin Layer; D006224:Cricetinae; D004975:Ethacridine / Q000494:pharmacology; D005047:Etoposide / Q000494:pharmacology; D006026:Glycoside Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D018922:HL-60 Cells; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D009447:Neuroblastoma; D010761:Phosphorus Radioisotopes; D014407:Tumor Cells, Cultured
D010761:Phosphorus Radioisotopes; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D005047:Etoposide; D006026:Glycoside Hydrolases; C022407:poly ADP-ribose glycohydrolase; D004975:Ethacridine