The Presence of Desulfo Xanthine Dehydrogenase in Purified and Crude Enzyme Preparations From Rat Liver

Arch Biochem Biophys. 1986 Jun;247(2):254-60. doi: 10.1016/0003-9861(86)90582-5.

Abstract

Crude and purified xanthine dehydrogenase preparations from rat liver were examined for the existence of a naturally occurring inactive form. Reduction of the purified enzyme by xanthine under anaerobic conditions proceeded in two phases. The enzyme was inactivated by cyanide, which caused the release of a sulfur atom from the molybdenum center as thiocyanate. The amount of thiocyanate released was almost in parallel with the initial specific activity. The active and inactive enzymes could be resolved by affinity chromatography on Sepharose 4B/folate gel. These results provided evidence that the purified enzyme preparation from rat liver contained an inactive form. A method for the determination of the active and inactive enzymes in crude enzyme preparations from rat liver was devised based on the fact that only active enzyme could react with [14C]allopurinol and both active and inactive enzymes could be immunoprecipitated quantitatively by excess specific antibody to xanthine dehydrogenase. The amount of [14C]alloxanthine (derived from [14C]allopurinol) bound to the active sulfo enzyme in crude rat liver extracts was about 0.5 mol/mol of FAD. As this content is closely similar to that in the purified enzyme, these results suggest the existence of an inactive desulfo form in vivo.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Allopurinol / pharmacology
  • Animals
  • Chromatography, Affinity
  • Electrophoresis, Disc
  • Ketone Oxidoreductases / isolation & purification*
  • Liver / enzymology*
  • Oxidation-Reduction
  • Potassium Cyanide / pharmacology
  • Rats
  • Rats, Inbred Strains
  • Spectrophotometry
  • Xanthine Dehydrogenase / antagonists & inhibitors
  • Xanthine Dehydrogenase / isolation & purification*

Substances

  • Allopurinol
  • desulfo xanthine dehydrogenase
  • Xanthine Dehydrogenase
  • Ketone Oxidoreductases
  • Potassium Cyanide