Studies on protein poly(ADP-ribosylation) using high resolution gel electrophoresis.
Analysis of poly(ADP-ribose) synthesized in cellular lysates or in isolated nuclei on 100-cm-long thin gels of 20% polyacrylamide, 2.5 M urea permits determination of the exact size of poly(ADP-ribose) molecules using labeled oligonucleotides as molecular weight markers. The size and concentration of poly(ADP-ribose) molecules increase at time intervals during its synthesis. Differences in the concentration of poly(ADP-ribose) size classes among cell lines are also shown. Inhibition of poly(ADP-ribose) degradation by ethacridine that directly interacts with the polymer and inhibits its hydrolysis by poly(ADP-ribose) glycohydrolase shows a dramatic increase in both polymer size and concentration. Use of alkaline conditions for the hydrolysis of poly(ADP-ribose)-protein linkages reveals a specific shortening of all size classes of poly(ADP-ribose) compared with its size in preparations obtained by extensive digestion of nuclei with nucleases, RNases, and proteases.
Boulikas
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Linus Pauling Institute of Science and Medicine, Palo Alto, California 94306.
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D000242:Cyclic AMP / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004975:Ethacridine / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D051379:Mice; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009702:Nucleoside Diphosphate Sugars / Q000096:biosynthesis*; D011064:Poly Adenosine Diphosphate Ribose / Q000096:biosynthesis* / Q000302:isolation & purification; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism
D009702:Nucleoside Diphosphate Sugars; D011506:Proteins; D009243:NAD; D011064:Poly Adenosine Diphosphate Ribose; D000242:Cyclic AMP; D004975:Ethacridine