Poly(ADP-ribosyl)ation of terminal deoxynucleotidyl transferase in vitro.
The activity of purified bovine thymus terminal deoxynucleotidyl transferase was markedly inhibited when the enzyme was incubated in a poly(ADP-ribose)-synthesizing system containing purified bovine thymus poly(ADP-ribose) polymerase, NAD+, Mg2+ and DNA. All of these four components were indispensable for the inhibition. The inhibitors of poly(ADP-ribose) polymerase counteracted the observed inhibition of the transferase. Under a Mg2+-depleted and acceptor-dependent ADP-ribosylating reaction condition [Tanaka, Y., Hashida, T., Yoshihara, H. and Yoshihara, K. (1979) J. Biol. Chem. 254, 12433-12438], the addition of terminal transferase to the reaction mixture stimulated the enzyme reaction in a dose-dependent manner, suggesting that the transferase is functioning as an acceptor for ADP-ribose. Electrophoretic analyses of the reaction products clearly indicated that the transferase molecule itself was oligo (ADP-ribosyl)ated. When the product was further incubated in the Mg2+-fortified reaction mixture, the activity of terminal transferase markedly decreased with increase in the apparent molecular size of the enzyme, indicating that an extensive elongation of poly(ADP-ribose) bound to the transferase is essential for the observed inhibition. Free poly(ADP-ribose) and the polymer bound to poly(ADP-ribose) polymerase were ineffective on the activity of the transferase. All of these results indicate that the observed inhibition of terminal transferase is caused by the poly(ADP-ribosyl)ation of the transferase itself.
Tanaka
Y
Y
Ito
K
K
Yoshihara
K
K
Kamiya
T
T
D000818:Animals; D002417:Cattle; D003101:Collodion; D004253:DNA Nucleotidylexotransferase / Q000378:metabolism*; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007120:Immunochemistry; D066298:In Vitro Techniques; D008274:Magnesium / Q000502:physiology; D009243:NAD / Q000378:metabolism; D009702:Nucleoside Diphosphate Sugars / Q000378:metabolism*; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D013950:Thymus Gland / Q000201:enzymology
D009702:Nucleoside Diphosphate Sugars; D009243:NAD; D011064:Poly Adenosine Diphosphate Ribose; D003101:Collodion; D011065:Poly(ADP-ribose) Polymerases; D004254:DNA Nucleotidyltransferases; D004253:DNA Nucleotidylexotransferase; D008274:Magnesium