Analysis of poly(ADP-ribose) glycohydrolase activity in nuclear extracts from mammalian cells

Rosa Bernardi, Laura Rossi, Guy G. Poirier, A. Ivana Scovassi

Research output: Contribution to journalArticle

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.

Original languageEnglish
Pages (from-to)60-68
Number of pages9
JournalBiochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Volume1338
Issue number1
DOIs
Publication statusPublished - Mar 7 1997

Keywords

  • ADP-ribose
  • Apoptosis
  • Autoradiography, P-
  • Catabolism
  • Chromatography, thin-layer
  • HL60 cell, etoposide-treated
  • Poly(ADP-ribose) glycohydrolase

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Structural Biology
  • Biophysics

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