Multiple autopoly(ADP-ribosyl)ation of rat liver poly(ADP-ribose) synthetase. Mode of modification and properties of automodified synthetase.

Abstract

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.Poly(ADP-ribose) synthetase is a chromatin-bound enzyme which synthesizes a protein-bound homopolymer, poly(ADPribose), from NAD (1-3).Various nuclear proteins, including histones and nonhistone proteins, have been reported as acceptors of this polymer in isolated nuclei (1-7) and in vivo (3, 7, 8).Recently, a branch structure of poly(ADP-ribose) was demonstrated by Miwa et al. (9).The enzyme responsible for the branching has not, yet been identified.Poly(ADP-ribose) synthetase has been purified to apparent homogeneity from various sources (10-15).Yoshihara et al. (16) and others (15, 17) reported that the purified synthetase