Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
Blood. 2011 Mar 10;117(10):2967-74. doi: 10.1182/blood-2010-08-304303. Epub 2011 Jan 14.
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.
我们描述了一种用于研究维生素 K 循环酶的基于细胞的测定法。一种由因子 IX 的 gla 结构域（氨基酸 1-46）和蛋白 C 的残基 47-420 组成的报告蛋白在 HEK293 和 AV12 细胞中稳定表达。当用维生素 K 或维生素 K 环氧化物（KO）喂养时，这两种细胞系均分泌羧化的报告蛋白。然而，当用华法林存在时，这两种细胞系都没有羧化报告蛋白。华法林存在时，维生素 K 挽救了 HEK293 细胞中的羧化作用，但在 AV12 细胞中却没有。二苯并恶唑，一种 NAD(P)H 依赖性醌氧化还原酶 1（NQO1）抑制剂，在这两种细胞系中均表现出类似于华法林的行为。当用华法林存在时，用 KO 喂养时，对维生素 K 具有抗性的维生素 K 环氧化物还原酶（VKOR-Y139F）支持 HEK293 细胞中的羧化作用，但在 AV12 细胞中却没有。这些结果表明：（1）我们的细胞系统是研究维生素 K 循环的良好模型，（2）将维生素 K 还原为氢醌（KH₂）的华法林抗性酶可能不是 NQO1，（3）似乎存在一种除 VKOR 以外的对华法林敏感的酶，可将维生素 K 还原为 KH₂，（4）VKOR 的主要功能是将 KO 还原为维生素 K。
