Functional study of the vitamin K cycle in mammalian cells.
We describe a cell-based assay for studying vitamin K-cycle enzymes. A reporter protein consisting of the gla domain of factor IX (amino acids 1-46) and residues 47-420 of protein C was stably expressed in HEK293 and AV12 cells. Both cell lines secrete carboxylated reporter when fed vitamin K or vitamin K epoxide (KO). However, neither cell line carboxylated the reporter when fed KO in the presence of warfarin. In the presence of warfarin, vitamin K rescued carboxylation in HEK293 cells but not in AV12 cells. Dicoumarol, an NAD(P)H-dependent quinone oxidoreductase 1 (NQO1) inhibitor, behaved similarly to warfarin in both cell lines. Warfarin-resistant vitamin K epoxide reductase (VKOR-Y139F) supported carboxylation in HEK293 cells when fed KO in the presence of warfarin, but it did not in AV12 cells. These results suggest the following: (1) our cell system is a good model for studying the vitamin K cycle, (2) the warfarin-resistant enzyme reducing vitamin K to hydroquinone (KH₂) is probably not NQO1, (3) there appears to be a warfarin-sensitive enzyme other than VKOR that reduces vitamin K to KH₂, and (4) the primary function of VKOR is the reduction of KO to vitamin K.
Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
Jian-Ke
JK
Tie
Da-Yun
DY
Jin
David L
DL
Straight
Darrel W
DW
Stafford
D000925:Anticoagulants / Q000494:pharmacology; D002460:Cell Line; D019411:Clinical Laboratory Techniques; D057809:HEK293 Cells; D006801:Humans; D006899:Mixed Function Oxygenases / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014812:Vitamin K / Q000378:metabolism*; D010837:Vitamin K 1 / Q000031:analogs & derivatives / Q000378:metabolism; D064417:Vitamin K Epoxide Reductases; D014859:Warfarin / Q000494:pharmacology
D000925:Anticoagulants; D014812:Vitamin K; C016186:vitamin K1 oxide; D014859:Warfarin; D010837:Vitamin K 1; D006899:Mixed Function Oxygenases; D064417:Vitamin K Epoxide Reductases
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