A method for determining oligo- and poly(ADP-ribosyl)ated enzymes and proteins in vitro.
A new method to determine oligo- and poly(ADP-ribosyl)ated enzymes and proteins in vitro has been developed. This method is based on the facts that in Mg2+-depleted condition automodification of poly(ADP-ribose)polymerase is minimized and exogenously added acceptor protein is oligo(ADP-ribosyl)ated predominantly, and in Mg2+-fortified conditions the exogenous acceptor can be poly(ADP-ribosyl)ated. When 13 proteins, including several enzymes, were subjected to this system, dimeric bovine seminal RNase and micrococcal nuclease were found to be oligo(ADP-ribosyl)ated under Mg2+-depleted conditions but their activity was unchanged. Under Mg2+-fortified conditions however, the RNase was deactivated concomitantly with its extensive poly(ADP-ribosyl)ation. When dimeric bovine seminal RNase was monomerized in advance by treatment with dithiothreitol and urea, the enzyme lost ADP-ribose-accepting ability in spite of a significant residual enzyme activity. As used here successfully, the Mg2+-depleted and Mg2+-fortified ADP-ribosylation and subsequent chromatographic analysis of various proteins and enzymes might be an useful method for proving their oligo- and poly(ADP-ribosyl)ation.
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D003851:Deoxyribonucleases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004798:Enzymes / Q000378:metabolism*; D008274:Magnesium; D008297:Male; D008836:Micrococcal Nuclease / Q000378:metabolism; D009702:Nucleoside Diphosphate Sugars / Q000378:metabolism*; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism; D012661:Semen / Q000201:enzymology
D004798:Enzymes; D009702:Nucleoside Diphosphate Sugars; D011506:Proteins; D000246:Adenosine Diphosphate Ribose; D011064:Poly Adenosine Diphosphate Ribose; D003851:Deoxyribonucleases; D012260:Ribonucleases; D008836:Micrococcal Nuclease; D008274:Magnesium