Multiple Autopoly ( ADP - ribosy 1 ) ation of Rat Liver Poly ( ADP - ribose ) Synthetase MODE

Poly(ADP4bose) synthetase of rat liver was found to catalyze automodification on multiple sites. When the synthetase was incubated with 2.4 p~ NAD for 20 s in the presence of DNA, more than 90% of ADP ribose incorporated into acid-insoluble material co-migrated with the synthetase (Mr = 108,000) upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incubation for longer times or at higher NAD concentrations led to the increase in the apparent molecular weight, up to >500,000, of the poly(ADP-ribosyl) enzyme. The enzyme-bound polymers had branch structures at a frequency of once every 50 ADP-ribose residues. After correction for branching, the enzyme was estimated to bind as many as 15 polymers of the average chain length of 80 or more ADP-ribose units. The poly(ADPribose)-enzyme linkage was labile in 0.1 N NaOH and 2 M NHzOH (pH 7.0) at 26 “C, indicating a similar type of ester bond as in ADP-ribosyl histones. The automodified enzyme was less active than the unmodified enzyme; the K,,, value for NAD gradually increased and V, decreased as the modification proceeded. During the automodification of the synthetase, little, if any, free oligomers and polymers were produced. The automodified enzyme isolated by gel filtration did not catalyze a transfer of its polymers to histone H1 in the presence or absence of NAD, although the automodified enzyme retained the activity to transfer ADP-ribose from NAD to the histone. Pulse-chase experiments supported this view that the polymers were not transferred from the enzyme to histone HI. These results suggested that the poly(ADP-ribosyl) enzyme was not an intermediate in poly(ADP-ribosy1)ation of other proteins.