Characterization and purification of the vitamin K-1 2,3 epoxide reductasesystem from rat liver

The enzyme vitamin K-1 2,3 epoxide reductase is responsible for converting vitamin K-1 2,3 epoxide to vitamin K-1 quinone thus completing the vitamin K cycle. The enzyme is also the target of inhibition by the oral anticoagul ant, R,S-warfarin. Purification of this protein would enable the interactio n of the inhibitor with its target to be elucidated. To date a single prote in possessing vitamin K-1 2,3 epoxide reductase activity and binding R,S-wa rfarin has yet to be purified to homogeneity, but recent studies have indic ated that the enzyme is in fact at least two interacting protei ns. We repo rt on the attempted purification of the vitamin K-1 2,3 epoxide reductase c omplex from rat liver microsomes by ion exchange and size exclusion chromat ography techniques. The intact system consisted of a warfarin-binding facto r, which possessed no vitamin K-1 2,3 epoxide reductase activity and a cata lytic protein. This catalytic protein was purified 327-fold and was insensi tive to R,S-warfarin inhibition at concentrations up to 5 mM. The addition of the S-200 size exclusion chromatography fraction containing the inhibito r-binding factor resulted in the return of R,S-warfarin inhibition. Thus, t o function normally, the rat liver endo plasm ic reticulum vita min K-1 2,3 epoxide reductase system requires the association of two components, one w ith catalytic activity for the conversion of the epoxide to the quinone and the second, the inhibitor binding factor. This latter enzyme forms the thi ol-disulphide redox centre that in the oxidized form binds R,S-warfarin.