Clarification of the conversion mechanism of vitamin K analogues to menaquinone-4, a potent hormone-like substance, in the rat tissues.

菜籽酮-4(MK-4)是维生素K_2(MK-n或K2)的一种天然存在形式，主要由微生物合成，但最近的研究表明，MK-4也可由其他维生素K类似物在大鼠各种组织中合成，也可能在其他哺乳动物组织中合成。根据我们和其他美国和荷兰研究人员最近的研究，MK-4在大鼠的睾丸、肺、脑、颌下腺、心、脾、肝、胰腺和肾脏等组织中的分布比其他VK类似物丰富。最近的研究结果还表明，MK-4是一种强有力的大鼠骨形成促进剂，因此MK-4被认为是一种强有力的激素样物质。我们利用大鼠的各种组织建立了MK-4的体外形成系统。该反应体系的底物为维生素K_3(叶二酮或维生素K_3)或维生素K_1(叶醌或维生素K_1)作为萘醌环的来源，香叶基香叶基-二磷酸a…S更是分别为MK-4的异戊二烯基侧链来源。取大鼠肾匀浆900g，磷酸盐缓冲液(pH 7.4)上清液作为粗酶溶液。实验表明，在VKS环氧化物和VKS还原酶抑制剂华法林的存在下，VK3产生的MK-4受到剂量相关的抑制。这种抑制作用可通过加入还原剂二硫苏糖醇(DTT)完全恢复。这使得我们认为VK还原酶参与了VK3向MK-4的转化反应，VK3-H_2(VK3的还原形式)和MK-4-H_2(MK-4的还原形式)可能作为中间产物参与了反应过程。对这一关键酶的纯化试验正在进行中，我们观察到从微粒体或线粒体膜部分解离使该酶更加不稳定(活性降低)。我们已经部分地阐明了VKS转化为MK-4的机制，并获得了有用的信息来纯化该酶(暂定名称为[MK-4合成酶])。要了解MK-4在大鼠不同组织中积累的生理作用，还需要进一步的研究。较少

Menaquinone-4 (MK-4) is one of naturally occurring form of vitmin K_2 (MK-n or K2) which is majorly synthesized in the microorganisms, but recently it has been shown that MK-4 is also synthesized from other vitamin K analogues in various tissues of rat and possibly in other mammalian tissues. According to recent investigations undertaken by us and other American and Dutch researchers, MK-4 abundantly distributes than other VK analogues to various tissues in the rats, such as testis, lung, brain, submandibullar gland, heart, spleen, liver, pancreas, and kidney. Recent results also showed that MK-4 is one of potent accelerator of bone formation in the rats, so MK-4 has been thought to be a potent hormone-like substance.We have newly established an MK-4 formation system in vitro using various tissues of the rats. Substrates for this reaction system are vitamin K_3 (menadione or VK3) or vitamin K_1 (phylloquinone or VK1) as a source for naphthoquinone ring, and geranylgeranyl-diphosphate a … More s a source for isoprenyl side-chain of MK-4, respectively. Supernatant fraction of 900 g of rat kidney homogenate with phophate buffer (pH 7.4) was used as crude enzyme solution. The present experiment undertaken for 2 years revealed that MK-4 production from VK3 was inhibited in a dose-related manner under the presence of warfarin that is an inhibitor for VKs epoxide and VKs reductases. This inhibition was recovered completely by an addition of reducing agent, di-thiothreitol (DTT). This lead us to consider that VK reductase participate in the conversion reaction from VK3 to MK-4, and that VK3-H_2 (reduced form of VK3) and MK-4-H_2 (reduced form of MK-4) might be involved in the reaction pathway as an intermediates. Purification trials of this key enzyme is now going on, and we are observing that the dissociation from the microsomal or mitochondrial membrane fraction made this enzyme much more unstable (less active).We have partly clarified the conversion mechanism of VKs into MK-4 much more clearly than before, and got useful information to purify this enzyme ([MK-4 synthase] as a tentative name). Further investigations are necessary to understand the physiological role of accumulated MK-4 in various tissues in the rat. Less