Purification and characterization of human lymphoid poly(adenosine diphosphate ribose) polymerase.
Poly(ADP-ribose) polymerase has been purified 12 000-fold from human tonsils with an 83% recovery of enzymatic activity relative to that of the initial homogenate. The specific activity of the purified enzyme is 862 units/mg of protein. The isolated protein has a molecular weight of approximately 116 000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent Km for NAD+ is estimated to be 185 microM at pH 8.0 and 37 degrees C. The purified enzyme has an absolute requirement for exogenous DNA for catalytic activity, and the reaction is enhanced by the addition of purified histone H1. The enzyme does not require magnesium or other divalent cations for activity. Enzyme activity is inhibited by p-(hydroxymercuri)benzoate and N-ethylmaleimide. Thymidine, theophylline, nicotinamide, and 5-methylnicotinamide markedly inhibit enzyme activity whereas ADP-ribose, 3',5'-cAMP, and sodium fluoride have a minimal effect on enzyme activity. Autoradiograms of labeled products of the reaction catalyzed by the purified enzyme at different concentrations of NAD+ and at different incubation times show that at low concentrations of NAD+ and after short incubations, poly(ADP-ribosyl)ation of the enzyme occurs preferentially over that of histone H1; at higher concentrations of NAD+ or after longer incubations, poly(ADP-ribosyl)ation of histone H1 is increased.
D002413:Cations, Divalent / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006657:Histones / Q000494:pharmacology; D006801:Humans; D008970:Molecular Weight; D009244:NAD+ Nucleosidase / Q000302:isolation & purification*; D014066:Palatine Tonsil / Q000201:enzymology; D011065:Poly(ADP-ribose) Polymerases / Q000302:isolation & purification* / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D006657:Histones; D013439:Sulfhydryl Reagents; D011065:Poly(ADP-ribose) Polymerases; D009244:NAD+ Nucleosidase