PURIFICATION AND BIOCHEMICAL-CHARACTERIZATION OF A POLY(ADP-RIBOSE) POLYMERASE-LIKE ENZYME FROM THE THERMOPHILIC ARCHAEON SULFOLOBUS-SOLFATARICUS

A poly(ADP-ribose) polymerase-like enzyme, detected in a crude homogen ate from Sulfolobus solfataricus by means of activity and immunoblot a nalyses, was purified to electrophoretic homogeneity by a rapid proced ure including two sequential affinity chromatographies, on NAD(+)-agar ose and DNA-Sepharose. The latter column selected specifically the pol y(ADP-ribosyl)ating enzyme with a 17% recovery of enzymic activity and a purification of more than 15 000-fold. The molecular mass (54-55 kD a) assessed by SDS/PAGE and immunoblot was definitely lower than that determined for the corresponding eukaryotic protein. The enzyme was pr oved to be thermophilic, with a temperature optimum of approx. 80 degr ees C, and thermostable, with a half-life of 204 min at 80 degrees C, in good agreement with the requirements of a thermozyme. It displayed a K-m towards NAD(+) of 154 +/- 50 mu M; in the pH range 6.5-10.0 the activity values were similar, not showing a real optimum pH. The enzym e was able to bind homologous DNA, as evidenced by the ethidium bromid e 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 re producing that obtained by the enzymic digestion of the rat testis pol y(ADP-ribose). These results strongly suggest that the activities of t he purified enzyme include the elongation step.