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

本研究首次证实MK-4是由各种组织（睾丸、肺、脑、下颌下腺、心脏、脾、肝、胰腺和肾）中的VK_1形成的同源系统，如我们先前描述的和其他研究人员提出的体内代谢（Suttie J.W.et at.和Thijssen et at.）。本实验还表明，组织同源因子是MK-4形成所必需的，并且所使用的组织来源蛋白可能对应于转化酶，存在剂量关系。匀浆的分级分离也进行了，并观察到最高的活性在微粒体部分，其次是线粒体部分，但其他馏分几乎没有活动。结合用TritonX-100增溶膜的实验结果，推测该转化酶可能为膜结合型，但仍需进一步研究。就底物特异性而言，当使用GGPP作为异戊二烯基侧链时，VK_1作为萘醌环的情况下比VK_3具有更高的活性，当使用VK_1作为萘醌环底物时，GGPP的活性也高于任何其他异戊二烯基侧链。使用[8-环]-^<14>C-VK_1底物的实验结果表明，VK类似物如还原-MK-4、VK^3、还原的VK^1和MK-4氧化物可能是最终形成MK-4的中间产物的候选物。我们通过改进提取方法和合成在常规实验室环境中被认为不稳定的还原形式的VK，在世界上首次获得了这些结果。

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.