Poly(ADP-ribose) synthase is the major endogenous nonhistone acceptor for poly(ADP-ribose) in alkylated rat hepatoma cells.
The endogenous poly(ADP-ribosyl)--nonhistone protein conjugates were isolated from dimethyl-sulfate-treated rat hepatoma AH 7974 cells using aminophenylboronic-acid--agarose chromatography. Seven major components could be discerned on dodecyl sulfate gels (molecular mass 43, 60, 66, 86, 100, 110 and 170 kDa) while control cells indicated only slight staining at above 200 kDa. The most abundant conjugate formed in response to alkylation damage was further purified using preparative gel electrophoresis and identified on the basis of its intrinsic enzymic activity as automodified poly(APD-ribose) synthase. In addition, topoisomerase I activity was found associated with a 60-kDa peptide. ADP-ribosylated endonuclease and actin were not detect-able. The purified conjugate fraction contained maximally 8.8 nmol/mg ADP-ribose and 7.9 nmol/mg oligo(ADP-ribose) with a mean chain length of 2.3 residues. The modifying (ADP-ribosyl)n groups were attached to its acceptors by a hydroxylamine-insensitive bond and had practically no effect on the DNA affinity of either poly(ADP-ribose) synthase or topoisomerase I.
D000477:Alkylating Agents / Q000494:pharmacology*; D000478:Alkylation; D000818:Animals; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000302:isolation & purification / Q000378:metabolism*; D006657:Histones / Q000378:metabolism; D007700:Kinetics; D008114:Liver Neoplasms, Experimental / Q000201:enzymology*; D009702:Nucleoside Diphosphate Sugars / Q000378:metabolism*; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D051381:Rats; D013463:Sulfuric Acid Esters / Q000494:pharmacology*; D013464:Sulfuric Acids / Q000494:pharmacology*
D000477:Alkylating Agents; D002868:Chromosomal Proteins, Non-Histone; D006657:Histones; D009702:Nucleoside Diphosphate Sugars; D013463:Sulfuric Acid Esters; D013464:Sulfuric Acids; D011064:Poly Adenosine Diphosphate Ribose; D011065:Poly(ADP-ribose) Polymerases; C007482:dimethyl sulfate