Ca2+, Mg2+-dependent endonuclease and ADP-ribosylation.
The molecular mechanism of the inhibition of Ca2+, Mg2+-dependent endonuclease by ADP-ribosylation was studied by using purified bull seminal plasma Ca2+, Mg2+-dependent endonuclease, endonuclease-stimulating proteins, and poly-(ADP-ribose) polymerase. The activity of an essentially homogeneous preparation of the endonuclease was markedly suppressed by its preincubation with NAD+, poly-(ADP-ribose) polymerase, DNA, and Mg2+. These four components of the incubation mixture were all essential for the suppression of the activity. Analyses of the initial and the chased reaction product by Sephadex G-100 column chromatography and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis revealed that Ca2+, Mg2+-dependent endonuclease was ADP-ribosylated during the incubation and its activity was markedly inhibited by the elongation of the ADP-ribose polymer covalently attached to the endonuclease. When the suppressed enzymes were mildly treated with an alkaline pH of 10.0, the activity was restored almost to the level of the unmodified control sample. These facts indicate that the linkage between the enzyme and poly(ADP-ribose) is hydrolyzed at this pH, and that the liberated polymer itself does not appreciably affect the endonuclease activity. These results also suggest that an electric repulsion between negative charges on DNA and poly(ADP-ribose) attached to Ca2+, Mg2+-dependent endonuclease is the basis for the observed suppression of the enzyme by ADP-ribosylation. Though histone H2B and H1 are shown to be as good endonuclease-stimulators (1) as they are good acceptors of ADP-ribose in poly(ADP-ribose) polymerase reaction (2), ADP-ribosylation of these two proteins did not affect their endonuclease-stimulating ability appreciably, at least under the conditions used.
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D004720:Endonucleases / Q000032:analysis / Q000378:metabolism*; D006657:Histones / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism*; D008297:Male; D009702:Nucleoside Diphosphate Sugars / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011485:Protein Binding; D012661:Semen / Q000201:enzymology
D006657:Histones; D009702:Nucleoside Diphosphate Sugars; D000246:Adenosine Diphosphate Ribose; D011065:Poly(ADP-ribose) Polymerases; D004720:Endonucleases; D008274:Magnesium; D002118:Calcium