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.