Kawamoto K, Uchida K
Shionogi and Co., Ltd., Osaka, Japan.
Jpn J Pharmacol. 1989 Jun;50(2):159-65. doi: 10.1254/jjp.50.159.
NADH-dependent vitamin K reductase activity in rat liver microsomes was measured by detecting the amount of the reduced form of vitamin K from the oxidized form of the vitamin. The enzyme activity was not detected when intact microsomes were employed as the enzyme source, but the solubilization of the microsomal enzyme with 1.5% Triton X-100 caused a development of the activity. Although the enzyme activity decreased gradually with time after the solubilization, the enzyme was stabilized by the addition of 20% glycerol and 2 mM vitamin C. Some optimal assay conditions for the vitamin K reductase were determined using the solubilized enzyme, and the standard assay method is described. Vitamin K reductase activity was not affected by warfarin and N-ethylmaleimide (NEM), but pyridoxal-5-phosphate (PAL-P) inhibited the activity, especially when microsomes were preincubated with PAL-P. The enzyme activity was not inhibited by N-methyltetrazolethiol (NMTT) and NMTT-containing antibiotics, suggesting that the hypoprothrombinemia caused by beta-lactam antibiotics was not due to the inhibition of NADH-dependent vitamin K reductase.
通过检测维生素K从氧化形式还原后的量来测定大鼠肝脏微粒体中依赖烟酰胺腺嘌呤二核苷酸（NADH）的维生素K还原酶活性。当完整的微粒体作为酶源时未检测到酶活性，但用1.5% Triton X-100溶解微粒体酶可使活性显现。尽管酶活性在溶解后随时间逐渐降低，但通过添加20%甘油和2 mM维生素C可使酶稳定。使用溶解后的酶确定了维生素K还原酶的一些最佳测定条件，并描述了标准测定方法。维生素K还原酶活性不受华法林和N-乙基马来酰亚胺（NEM）影响，但磷酸吡哆醛（PAL-P）抑制该活性，尤其是当微粒体与PAL-P预孵育时。该酶活性不受N-甲基四氮唑硫醇（NMTT）和含NMTT的抗生素抑制，这表明β-内酰胺类抗生素引起的低凝血酶原血症并非由于依赖NADH的维生素K还原酶受到抑制。
