Purification and biochemical characterization of a poly(ADP-ribose) polymerase-like enzyme from the thermophilic archaeon Sulfolobus solfataricus

Biochem J. 1998 Oct 15;335 ( Pt 2)(Pt 2):441-7. doi: 10.1042/bj3350441.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Archaeal Proteins / chemistry
  • Archaeal Proteins / isolation & purification*
  • Archaeal Proteins / metabolism*
  • Chromatography, Affinity
  • Chromatography, High Pressure Liquid / methods
  • DNA / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Stability
  • Hot Temperature
  • Hydrogen-Ion Concentration
  • Kinetics
  • Molecular Weight
  • NAD / metabolism
  • Phosphodiesterase I
  • Phosphoric Diester Hydrolases / chemistry
  • Phosphoric Diester Hydrolases / metabolism
  • Poly(ADP-ribose) Polymerases / chemistry
  • Poly(ADP-ribose) Polymerases / isolation & purification
  • Poly(ADP-ribose) Polymerases / metabolism*
  • Rats
  • Sulfolobus / chemistry*

Substances

  • Archaeal Proteins
  • NAD
  • DNA
  • Poly(ADP-ribose) Polymerases
  • Phosphoric Diester Hydrolases
  • Phosphodiesterase I