ADP-ribosylation of nuclear proteins in vivo. Identification of histone H2B as a major acceptor for mono- and poly(ADP-ribose) in dimethyl sulfate-treated hepatoma AH 7974 cells.
Nuclear mono- and poly(ADP-ribosyl) protein conjugates formed in living hepatoma AH 7974 cells in response to treatment with the alkylating agent dimethyl sulfate have been studied. They were isolated from the perchloric acid precipitate of freshly prepared nuclei in a relatively pure form and with an overall yield of more than 80%, utilizing aminophenylboronic acid-agarose chromatography. Exposure of the cells to 400 microM dimethyl sulfate led to a transient rise of ADP-ribosylated proteins. After 20 min, the level of endogenous poly(ADP-ribosyl) residues increased by a factor of 21, amounting to a final value of 772 +/- 57 pmol/mg of DNA while the mono(ADP-ribosyl) residues were raised to even higher concentrations (1864 pmol/mg of DNA), corresponding to a 12-fold stimulation as compared to untreated cells. As a result of dimethyl sulfate treatment, the amount of acceptor protein being modified by (ADP-ribose)n was elevated 15-fold, reaching a final proportion of 2.3 +/- 0.4% of total nuclear protein. The increase in (ADP-ribosyl)n-modified proteins was suppressed by benzamide, a potent inhibitor of poly(ADP-ribose) synthetase. More than half of the nuclear mono- and poly(ADP-ribosyl) residues were linked to histone H2B. The modifying residues could be removed from the major acceptor by treatment with 0.1 M NaOH, but not with neutral hydroxylamine. Minor amounts of other histones, especially of histone H4, were possibly also ADP-ribosylated under the stimulating effect of dimethyl sulfate. In addition, several nonhistone proteins with apparent molecular masses of 100-116 and 170 kDa were found to carry substantial amounts of mono- and poly(ADP-ribose).
D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006657:Histones / Q000378:metabolism*; D008114:Liver Neoplasms, Experimental / Q000378:metabolism*; D008970:Molecular Weight; D009702:Nucleoside Diphosphate Sugars / Q000378:metabolism*; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013463:Sulfuric Acid Esters / Q000494:pharmacology*; D013464:Sulfuric Acids / Q000494:pharmacology*
D001549:Benzamides; D006657:Histones; D009702:Nucleoside Diphosphate Sugars; D013463:Sulfuric Acid Esters; D013464:Sulfuric Acids; D011064:Poly Adenosine Diphosphate Ribose; C037689:benzamide; C007482:dimethyl sulfate