Mechanism of the inhibition of Ca2+, Mg2+-dependent endonuclease of bull seminal plasma induced by ADP-ribosylation.
The activity of purified Ca2+, Mg2+-dependent endonuclease was inhibited when the enzyme was incubated in a system containing poly(ADP-ribose) synthetase, NAD+, Mg2+, and DNA. All four ingredients were essential to mediate ADP-ribosylation and to demonstrate inhibition of the endonuclease. In the absence of Mg2+, ADP-ribose transferring activity of poly(ADP-ribose) synthetase was stimulated by the addition of purified endonuclease to the reaction mixture in a dose-dependent manner. Analysis of the reaction product showed that the endonuclease was ADP-ribosylated. The average chain length of the initial oligo(ADP-ribose) attached to the enzyme was about 5.9 residues. The oligomer was found to be extensively elongated during the chase experiment using unlabeled NAD+ and Mg2+. The present finding suggests that Mg2+ is essential for the extensive elongation of the oligo(ADP-ribose). The DNA-binding affinity of the modified endonuclease was significantly lower than that of unmodified enzyme. Also, free poly(ADP-ribose) was not an effective inhibitor of the endonuclease. These findings suggest that the observed inhibition of the endonuclease induced by ADP-ribosylation is probably due to an electrostatic repulsion between the substrate (DNA) and poly(ADP-ribose) covalently linked to the endonuclease. Histone H1 and H2B stimulated endonuclease activity and were acceptors of ADP-ribose; however, their capacity to stimulate endonuclease activity remained unchanged after ADP-ribosylation.
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D004706:Endodeoxyribonucleases / Q000037:antagonists & inhibitors*; D007700:Kinetics; D008297:Male; D009243:NAD / Q000378:metabolism; D009244:NAD+ Nucleosidase / Q000378:metabolism*; D009702:Nucleoside Diphosphate Sugars / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D011485:Protein Binding; D012661:Semen / Q000201:enzymology*; D014316:Tritium
D009702:Nucleoside Diphosphate Sugars; D009243:NAD; D014316:Tritium; D000246:Adenosine Diphosphate Ribose; D011065:Poly(ADP-ribose) Polymerases; D004706:Endodeoxyribonucleases; C029367:calcium magnesium dependent endodeoxyribonuclease; D009244:NAD+ Nucleosidase