|Budget Amount *help
¥2,400,000 (Direct Cost : ¥2,400,000)
Fiscal Year 1998 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1997 : ¥1,400,000 (Direct Cost : ¥1,400,000)
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.