Isolation and properties of an NAD- and guanidine-dependent ADP-ribosyltransferase from turkey erythrocytes.
An NAD- and guanidine-dependent ADP-ribosyltransferase has been purified more than 500,000-fold from turkey erythrocytes with an 18% yield. The enzyme in the 100,000 X g supernatant fraction was bound to phenyl-Sepharose, eluted with 50% propylene glycol, and further purified by sequential chromatographic steps on carboxymethylcellulose, NAD-agarose and concanavalin A-agarose. The transferase was specifically eluted from concanavalin A-agarose with alpha-methylmannoside. The enzymatic activity was extremely labile following the first purification step. Both propylene glycol and NaCl stabilized the transferase; significant increases in enzyme recovery were obtained by conducting the NAD- and concanavalin A-agarose chromatography in buffer containing propylene glycol. The purified protein exhibits one predominant protein band on SDS-polyacrylamide gels with an estimated molecular weight of 28,300. On Ultrogel AcA54 chromatography, single coincident peaks of ADP-ribosyltransferase activity and protein were observed. Enzyme activity was independent of DNA; the highly purified transferase was inhibited by thymidine, nicotinamide, and theophylline. The specific activity of the purified enzyme (350 mumol of ADP-ribose transferred from NAD to arginine methyl estermin-1mg-1) is comparable to that reported for purified NAD glycohydrolases and poly(ADP-ribosyl)transferases.
D000818:Animals; D004912:Erythrocytes / Q000201:enzymology*; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D009243:NAD; D009536:Niacinamide / Q000494:pharmacology; D009713:Nucleotidyltransferases / Q000097:blood* / Q000302:isolation & purification; D011065:Poly(ADP-ribose) Polymerases; D013806:Theophylline / Q000494:pharmacology; D013936:Thymidine / Q000494:pharmacology; D014422:Turkeys
D006146:Guanidines; D009243:NAD; D009536:Niacinamide; D013806:Theophylline; D011065:Poly(ADP-ribose) Polymerases; D009713:Nucleotidyltransferases; D013936:Thymidine