Purification and properties of poly(ADP-ribose) polymerase from lamb thymus.
Poly(ADP-ribose) polymerase was purified 2900-fold from lamb thymus with a recovery of 5%. Addition of exogenous DNA was essential for activity of the purified enzyme, and the reaction was stimulated by the addition of either a mixture of histones or purified histone H1. The enzyme is inhibited by sulfhydryl binding agents such as phenylmethanesulfonyl fluoride or N-ethylmaleimide. It does not require magnesium or other metal ion cofactors for activity. The enzyme migrated as a single polypeptide chain with an apparent molecular weight of 135 000 when gel electrophoresis was performed in the presence of sodium dodecyl sulfate. The apparent molecular weight was 175 000 when determined by gel filtration on Sepharose CL-6B-200. The isoelectric point was pH 9.6, and the pH optimum for activity was 8.6-8.8. The apparent Km for NAD+ was 160 microM at 37 degrees C. The activity of the purified polymerase was unaffected by the presence of ADP-ribose, 3',5'-cAMP, or NaF. Nicotinamide, 5-methyl-nicotinamide, theophylline, and thymidine markedly inhibited enzyme activity. Lamb thymus DNA, originally associated with the enzyme, was more effective than commercially obtained calf thymus DNA as an enzyme activator.
D000818:Animals; D002417:Cattle; D004247:DNA / Q000494:pharmacology; D004789:Enzyme Activation; D006657:Histones / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD; D009244:NAD+ Nucleosidase / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000302:isolation & purification / Q000378:metabolism*; D012756:Sheep; D013696:Temperature; D013950:Thymus Gland / Q000201:enzymology*
D006657:Histones; D009243:NAD; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases; D009244:NAD+ Nucleosidase