Kawamoto K, Touchi A, Sugeno K, Matsubara T
Shionogi Research Laboratories, Shionogi & Co., Ltd., Osaka, Japan.
Jpn J Pharmacol. 1988 Jun;47(2):169-78. doi: 10.1254/jjp.47.169.
Liver microsomal vitamin K epoxide reductase activity was determined by measuring the formation of menaquinone-4 from the substrate menaquinone-4 2,3-epoxide. The enzyme was active when dithiothreitol (DTT) was used as a reducing agent, and the activity increased gradually with increasing concentrations of DTT. Glutathione and cysteine also functioned as reductants, but these physiological reductants showed less than 15% of the activity detected with 0.5 mM DTT. Addition of various beta-lactam antibiotics to the assay mixture for vitamin K epoxide reductase caused a slight inhibition of the activity. N-Methyltetrazolethiol (NMTT) and other heterocyclic thiol compounds also inhibited the enzyme activity in vitro depending on their concentrations. Most of these antibiotics and heterocyclic thiol compounds inhibited the enzyme activity only 10-25% in the in vitro assay system even when higher concentrations were added (5-10 mM). Among the compounds tested, methyl-thiadiazolethiol was the only compound that caused 50% inhibition of the enzyme activity. NMTT-induced inhibition was diminished gradually by increasing DTT concentrations. Kinetic analysis of the inhibitory action of heterocyclic thiol compounds showed competitive inhibition against the reductant DTT and non-competitive inhibition against the substrate. On the other hand, warfarin, a typical anticoagulant, showed different patterns in the inhibitory action: non-competitive inhibition against DTT and mixed-type inhibition against the substrate.
通过测量底物维生素K4 2,3-环氧化物生成维生素K4来测定肝微粒体维生素K环氧化物还原酶的活性。当使用二硫苏糖醇（DTT）作为还原剂时，该酶具有活性，并且活性随着DTT浓度的增加而逐渐升高。谷胱甘肽和半胱氨酸也可作为还原剂，但这些生理性还原剂的活性不到0.5 mM DTT所检测活性的15%。向维生素K环氧化物还原酶的测定混合物中添加各种β-内酰胺抗生素会导致活性略有抑制。N-甲基四唑硫醇（NMTT）和其他杂环硫醇化合物在体外也会根据其浓度抑制该酶的活性。即使添加更高浓度（5-10 mM），在体外测定系统中，大多数这些抗生素和杂环硫醇化合物对酶活性的抑制也仅为10%-25%。在所测试的化合物中，甲基噻二唑硫醇是唯一能导致酶活性50%抑制的化合物。随着DTT浓度的增加，NMTT诱导的抑制作用逐渐减弱。杂环硫醇化合物抑制作用的动力学分析表明，其对还原剂DTT表现为竞争性抑制，对底物表现为非竞争性抑制。另一方面，典型的抗凝剂华法林在抑制作用上表现出不同的模式：对DTT为非竞争性抑制，对底物为混合型抑制。
