Characterization and Purification of the Vitamin K1 2,3 Epoxide Reductases System From Rat Liver

J Pharm Pharmacol. 2001 Apr;53(4):481-6. doi: 10.1211/0022357011775776.

Abstract

The enzyme vitamin K1 2,3 epoxide reductase is responsible for converting vitamin K1 2,3 epoxide to vitamin K1 quinone thus completing the vitamin K cycle. The enzyme is also the target of inhibition by the oral anticoagulant, R,S-warfarin. Purification of this protein would enable the interaction of the inhibitor with its target to be elucidated. To date a single protein possessing vitamin K1 2,3 epoxide reductase activity and binding R,S-warfarin has yet to be purified to homogeneity, but recent studies have indicated that the enzyme is in fact at least two interacting proteins. We report on the attempted purification of the vitamin K1 2,3 epoxide reductase complex from rat liver microsomes by ion exchange and size exclusion chromatography techniques. The intact system consisted of a warfarin-binding factor, which possessed no vitamin K1 2,3 epoxide reductase activity and a catalytic protein. This catalytic protein was purified 327-fold and was insensitive to R,S-warfarin inhibition at concentrations up to 5 mM. The addition of the S-200 size exclusion chromatography fraction containing the inhibitor-binding factor resulted in the return of R,S-warfarin inhibition. Thus, to function normally, the rat liver endoplasmic reticulum vitamin K1 2,3 epoxide reductase system requires the association of two components, one with catalytic activity for the conversion of the epoxide to the quinone and the second, the inhibitor binding factor. This latter enzyme forms the thiol-disulphide redox centre that in the oxidized form binds R,S-warfarin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Benzoquinones
  • Binding Sites
  • Catalysis
  • Chromatography, High Pressure Liquid
  • Endoplasmic Reticulum / enzymology
  • Microsomes, Liver / chemistry
  • Microsomes, Liver / enzymology
  • Mixed Function Oxygenases / isolation & purification*
  • Mixed Function Oxygenases / pharmacology
  • Oxidation-Reduction
  • Rats
  • Vitamin K 1 / analogs & derivatives*
  • Vitamin K 1 / metabolism*
  • Vitamin K Epoxide Reductases
  • Warfarin / pharmacokinetics

Substances

  • Benzoquinones
  • vitamin K1 oxide
  • quinone
  • Warfarin
  • Vitamin K 1
  • Mixed Function Oxygenases
  • Vitamin K Epoxide Reductases