Purification of a vitamin K epoxide reductase that catalyzes conversion of vitamin K 2,3-epoxide to 3-hydroxy-2-methyl-3-phytyl-2,3-dihydronaphthoquinone.
An enzyme from bovine liver microsomes that catalyzes the reduction of vitamin K 2,3-epoxide to 2- and 3-hydroxy-2-methyl-3-phytyl-2,3-dihydronaphthoquinone was purified 1152-fold to apparent homogeneity. Microsomes were solubilized with 3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and the enzyme was purified by chromatography on PBE-94 ion exchanger, hydroxylapatite, and DEAE-cellulose, and then gel filtration on Sephacryl S-200. The homogeneity of the final preparation was established by polyacrylamide slab gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weight of the native enzyme is 25,000 and that of denatured enzyme is 12,400, which suggests that the enzyme is a dimer with identical subunits. No chromophoric cofactors are associated with the enzyme. Dithiothreitol and CHAPS are essential for activity, but high concentrations of glycerol reduces the activity. The enzyme is not inhibited by warfarin, a potent inhibitor of the vitamin K epoxide reductase, which catalyzes the conversion of vitamin K 2,3-epoxide to vitamin K. Evidence is presented indicating that the purified enzyme is not simply a fragment of the warfarin-sensitive vitamin K epoxide reductase.
D000818:Animals; D002417:Cattle; D004229:Dithiothreitol / Q000494:pharmacology; D008862:Microsomes, Liver / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D011487:Protein Conformation; D014812:Vitamin K / Q000031:analogs & derivatives / Q000096:biosynthesis; D010837:Vitamin K 1 / Q000031:analogs & derivatives* / Q000378:metabolism; D064417:Vitamin K Epoxide Reductases; D014859:Warfarin / Q000494:pharmacology
D014812:Vitamin K; C016186:vitamin K1 oxide; D014859:Warfarin; C045267:3-hydroxy-2-methyl-3-phytyl-2,3-dihydronaphthoquinone; D010837:Vitamin K 1; D006899:Mixed Function Oxygenases; D064417:Vitamin K Epoxide Reductases; D004229:Dithiothreitol