Menaquinone-4, a potent anti-osteoporosis vitamin : Clarification of its intracellular distribution and new physiological function

本研究首次证实了MK-4是由VK_1在各种组织（睾丸、肺、脑、下颚腺、心脏、脾脏、肝脏、胰腺和肾脏）中均质化形成的，这可能是我们之前描述的和其他研究人员提出的体内代谢系统(Suttie J.W.et at。Thijssen等)。本实验还揭示了组织均质因子对MK-4的形成是必不可少的，并且使用的组织源蛋白可能与转化酶相对应，存在剂量关系。对匀浆也进行了分离，观察到微粒体部分活性最高，其次是线粒体部分，但其他部分几乎没有活性。结合膜增溶试剂Triton X-100的实验数据，认为该转化酶可能为膜相关型，但仍需进一步研究。在底物特异性方面，以GGPP为异丙烯基侧链时，以VK_1为萘醌环的活性高于以VK_3为萘醌环的活性；以VK_1为萘醌环底物时，以GGPP为萘醌环的活性高于其他异丙烯基侧链。使用[8-ring]-^<14>C-VK_1底物的实验结果表明，还原的MK-4、VK^3、还原的VK^1和MK-4氧化物等VK类似物可能是最终形成MK-4的中间产物。我们通过改进提取方法和合成在常规实验室环境下被认为不稳定的VK的还原形式，在世界上首次得到了这些结果。

It was confirmed for the first time by the present study that MK-4 is formed from VK_1 in various tissues (testis, lung, brain, submandibullar gland, heart, spleen, liver, pancreas, and kidney) homegenate system as likely in vivo metabolism described previously by us and suggested by other researchers (Suttie J.W.et at. and Thijssen et at.). The present experiment also revealed that the tissue homegenate factor is indispensable for MK-4 formation, and there was a dose-relationship of used tissue-originated protein that may correspond to the converting enzyme. The fractionation of the homogenate was also undertaken, and the highest activity was observed in the microsomal fraction, and followed by mitochondrial fraction, but the other fractions had almost no activity. Combined these results with the data from the experiment using membrane-solubilizing reagent, Triton X-100, it was suggested that the converting enzyme is probably membrane-associated type, though further studies are necessary. As for the substrate-specificity, the higher activities were observed in the case of VK_1 rather than VK_3 as naphthoquinone ring when GGPP was used as isoprenyl side chain, and also higher activity in the case of GGPP than any other isoprenyl side chains when VK_1 was used as naphthoquinone ring substrate.Results from the experiment using [8-ring]-^<14>C-VK_1 substrate indicate that the VK analogues such as reduced-MK-4, VK^3, reduced VK^1, and MK-4 oxide could be candidates for intermediates products for final MK-4 formation. We have got these results for the first time in the world by improving the extraction method and by synthesizing the reduced form of VK that has been thought to be unstable in a routine laboratory circumstance.