Purified vitamin K epoxide reductase alone is sufficient for conversion of vitamin K epoxide to vitamin K and vitamin K to vitamin KH2.

Abstract:

: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.

authors

Chu PH,Huang TY,Williams J,Stafford DW

doi

10.1073/pnas.0609401103

subject

Has Abstract

pub_date

2006-12-19 00:00:00

pages

19308-13

issue

51

eissn

0027-8424

issn

1091-6490

pii

0609401103

journal_volume

103

pub_type

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