Effect of ionic strength on chain elongation in ADP-ribosylation of various nucleases.
With the use of a reconstituted poly(ADP-ribosyl)ating enzyme system and three purified nucleases, micrococcal nuclease (MN), bull seminal RNase (BS RNase) and Ca2+, Mg2+-dependent endonuclease (BS DNase), as model acceptor proteins for ADP-ribose, the effect of ionic strength on the modification reaction was examined in detail. When these three nucleases were extensively poly(ADP-ribosyl)ated in this system at a low ionic strength (5 mM Tris), they were all inhibited by about 80% and the chain length of the polymer covalently bound to the nucleases was 13 to 23 ADP-ribose units. The observed inhibition was markedly prevented by increasing the ionic strength in the reaction mixture with a concomitant decrease in the polymer size bound to the nucleases. The NaCl concentrations required for decreasing the extent of the inhibition to half of the maximum were calculated to be 20, 50, and 100 mM for MN, BS RNase, and BS DNase, respectively. These values are similar to the NaCl concentrations required for decreasing the average chain lengths of the polymer to half, suggesting that the length of polymer is closely correlated to the extent of inhibition of these nucleases. DNA-binding affinities of these nucleases, expressed in terms of the NaCl concentrations required for eluting the enzymes from DNA-cellulose, were 140, 280, and 340 mM for MN, BS RNase, and BS DNase, respectively. Considering that maintainance of a ternary complex of poly(ADP-ribose) synthetase, acceptor and DNA may be essential for the modification reaction, the relatively strong salt effect observed in the modification of MN may be explained by its low DNA-binding affinity.
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Ohashi
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D001345:Autoradiography; D002118:Calcium / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D008836:Micrococcal Nuclease / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009702:Nucleoside Diphosphate Sugars / Q000378:metabolism*; D009994:Osmolar Concentration; D012260:Ribonucleases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology
D009702:Nucleoside Diphosphate Sugars; D000246:Adenosine Diphosphate Ribose; D012965:Sodium Chloride; D004247:DNA; D004706:Endodeoxyribonucleases; D012260:Ribonucleases; C029367:calcium magnesium dependent endodeoxyribonuclease; D008836:Micrococcal Nuclease; D008274:Magnesium; D002118:Calcium