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A poly(ADP-ribose) polymerase-like enzyme, detected in a crude homogenate from Sulfolobus solfataricus by means of activity and immunoblot analyses, was purified to electrophoretic homogeneity by a rapid procedure including two sequential affinity chromatographies, on NAD+-agarose and DNA-Sepharose. The latter column selected specifically the poly(ADP-ribosyl)ating enzyme with a 17% recovery of enzymic activity and a purification of more than 15000-fold. The molecular mass (54-55 kDa) assessed by SDS/PAGE and immunoblot was definitely lower than that determined for the corresponding eukaryotic protein. The enzyme was proved to be thermophilic, with a temperature optimum of approx. 80 degreesC, and thermostable, with a half-life of 204 min at 80 degreesC, in good agreement with the requirements of a thermozyme. It displayed a Km towards NAD+ of 154+/-50 microM; in the pH range 6.5-10.0 the activity values were similar, not showing a real optimum pH. The enzyme was able to bind homologous DNA, as evidenced by the ethidium bromide displacement assay. The product of the ADP-ribosylating reaction co-migrated with the short oligomers of ADP-ribose (less than 6 residues) from a eukaryotic source. Reverse-phase HPLC analysis of the products, after digestion with phosphodiesterase I, gave an elution profile reproducing that obtained by the enzymic digestion of the rat testis poly(ADP-ribose). These results strongly suggest that the activities of the purified enzyme include the elongation step.

The nuclear acceptor proteins for poly(ADP-ribose) were investigated in mouse liver and testis. In liver, histones are ribosylated preferentially, whereas in testis the major acceptors are non-histone proteins. An analysis of the purified testicular acceptor proteins suggests that they are high- and low-mobility-group-like proteins.