Purified vitamin K epoxide reductase alone is sufficient for conversion of vitamin K epoxide to vitamin K and vitamin K to vitamin KH2.
More than 21 million prescriptions for warfarin are written yearly in the U.S. Despite its importance, warfarin's target, vitamin K epoxide reductase (VKOR), has resisted purification since its identification in 1972. Here, we report its purification and reconstitution. HPC4, a calcium-specific antibody that recognizes a 12-aa tag, was used to purify and identify VKOR. Partial reconstitution is achieved on the column by washing with 0.4% dioleoylphosphatidylcholine/0.4% deoxycholate. Activity is completely recovered by dialysis against a buffer containing a reducing agent but lacking dioleoylphosphatidylcholine/deoxycholate. Removal of detergent from the eluted proteins apparently facilitates liposome formation. Purified recombinant VKOR with tag is approximately 21 kDa, as expected; fully active; and > 93% pure. The concentration of warfarin for 50% inhibition is the same for purified protein and microsomes. It has been reported that VKOR is a multisubunit enzyme. Our results, however, suggest that a single peptide can accomplish both the conversion of vitamin K epoxide to vitamin K and vitamin K to reduced vitamin K. This purification will allow further characterization of VKOR in relation to other components of the vitamin K cycle and should facilitate its structural determination.
Chu
Pei-Hsuan
PH
Department of Biology, University of North Carolina, Chapel Hill, NC 27599-3280, USA.
Huang
Teng-Yi
TY
Williams
Jason
J
Stafford
D W
DW
D016367:Baculoviridae; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D006899:Mixed Function Oxygenases / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D014812:Vitamin K / Q000096:biosynthesis*; D010837:Vitamin K 1 / Q000031:analogs & derivatives / Q000378:metabolism; D064417:Vitamin K Epoxide Reductases; D014859:Warfarin / Q000037:antagonists & inhibitors
D014812:Vitamin K; C016186:vitamin K1 oxide; D014859:Warfarin; D010837:Vitamin K 1; D006899:Mixed Function Oxygenases; D064417:Vitamin K Epoxide Reductases