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A molecular mechanism for genetic warfarin resistance in the rat
Author(s) -
Wallin Reidar,
Hutson Susan M.,
Cain Dean,
Sweatt Andrew,
Sane David C.
Publication year - 2001
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.01-0337fje
Subject(s) - recombinant dna , vitamin k epoxide reductase , warfarin , enzyme , biochemistry , chemistry , vitamin , in vitro , biology , microbiology and biotechnology , cytochrome p450 , gene , medicine , cyp2c9 , atrial fibrillation
ABSTRACT Warfarin targets vitamin K 2,3‐epoxide reductase (VKOR), the enzyme that produces reduced vitamin K, a required cofactor for γ‐carboxylation of vitamin K‐dependent proteins. To identify VKOR, we used 4′‐azido‐warfarin‐ 3 H‐alcohol as an affinity label. When added to a partially purified preparation of VKOR, two proteins were identified by mass spectrometry as calumenin and cytochrome B 5 . Rat calumenin was cloned and sequenced and the recombinant protein was produced. When added to an in vitro test system, the 47 kDa recombinant protein was found to inhibit VKOR activity and to protect the enzyme from warfarin inhibition. Calumenin was also shown to inhibit the overall activity of the complete vitamin K‐dependent γ‐carboxylation system. The results were repeated in COS‐1 cells overexpressing recombinant calumenin. By comparing calumenin mRNA levels in various tissues from normal rats and warfarin‐resistant rats, only the livers from resistant rats were different from normal rats by showing increased levels. Partially purified VKOR from resistant and normal rat livers showed no differences in Km‐values, specific activity, and sensitivity to warfarin. A novel model for genetic warfarin resistance in the rat is proposed, whereby the concentration of calumenin in liver determines resistance.