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 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.
Dipartimento di Chimica Organica e Biologica, Facoltà di Scienze, Università 'Federico II', Via Mezzocannone 16, 80134 Napoli, Italy. faraone@ds.unina.it
M R
MR
Faraone-Mennella
A
A
Gambacorta
B
B
Nicolaus
B
B
Farina
D000818:Animals; D019843:Archaeal Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D016944:Sulfolobus / Q000737:chemistry*
D019843:Archaeal Proteins; D009243:NAD; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I