Isolation of ADP-ribosyltransferase by affinity chromatography.
An affinity adsorbent for ADP-ribosyltransferase (EC 2.4.2.30) has been synthesized by coupling 3-aminobenzamide to Sepharose 4B. Using this material, ADP-ribosyltransferase from human placenta has been purified from crude extract to homogeneity within a few hours. The enzyme has an apparent Km for NAD+ of 52 microM. Its molecular mass is 115,000 as determined by gel electrophoresis. The enzyme is DNA dependent and stimulated by histone, its temperature optimum is at 25 degrees C, and its pH optimum is around pH 9. alpha-NAD+, thymidine, caffeine, theophylline, theobromine, 3-methoxybenzamide, and nicotinamide inhibit the enzyme. Purification of ADP-ribosyltransferases from horse, rat, and chicken liver was also achieved with the method described.
D000818:Animals; D001549:Benzamides; D002846:Chromatography, Affinity / Q000379:methods*; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008970:Molecular Weight; D009713:Nucleotidyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D010920:Placenta / Q000201:enzymology; D011065:Poly(ADP-ribose) Polymerases; D011247:Pregnancy; D012685:Sepharose
D001549:Benzamides; C025160:3-aminobenzamide; D012685:Sepharose; D011065:Poly(ADP-ribose) Polymerases; D009713:Nucleotidyltransferases