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Research of the physiological role of marine mollusks cross -linking enzymes and structures of its substrates.

研究海洋软体动物交联酶的生理作用及其底物结构。

基本信息

批准号：
17580177
负责人：
NOZAWA Hisanori
金额：
$ 2.41万
依托单位：
Hokkaido University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2007
项目状态：
已结题

项目摘要

Marine mollusk contains a transglutaminase (Tgase) which is activated by a contact with sea water or body fluid, suggesting it might be acted to stop bleeding by cross linking of proteins. To clarify the physiological role of Tease, identification of its intracellular substrates and common structural properties were investigatedThe main component of the intracellular substrate of scallop hemocyte, 45 kDa protein, was revealed to be Actin by amino-terminal sequences of BrCN-cleaved 15k and 12k fragments, YVAIQVLSL and GQKDSYVGDE, respectively.When carp myosin was incubated with Tgase and monodansylcadaverine MO, which is a fluorescent substrate of the enzyme, MDC was specifically incorporated in to S2 region of myosin heavy chain. B PLC analysis of the MDC-incorporated fragments derived from BrCN-cleavage and proteinase digestion showed that Gln(520) was the first cross-linking site in the myosin heavy chain. The Gln(520) was also identified to be the primary mss-linking site of Alaska pollack myosin. To compare the reactivity of scallop myosin heavy chain to those of fish ones, the complete amino acid sequence of scallop myosin heavy chain was determined by cDNA sequence analysis for the first time. The cross linking sites of scallop myosin were located in several Gln residues in the rod region containing Gln(520), suggesting the difference in the reactivity between substrate proteins.The intracellular substrates for squid gill Tgases were insoluble 190 kDa and 90 kDa proteins for Japanese common squid, and 190 kDa component for spear squid, respectively. Although the amino-terminal sequences of two BrCN-cleaved fragments of 190 kDa protein were determined to be AIDVE and ANTDEAL, respectively, no significant homology to the known proteins was detected. As the 200 kDa protein was also supposed to be a primary intracellular substrate for scallop hemocyte Tgase, these proteins were suggested to be common intracellular substrates for marine molluskan Tgases.

海洋软体动物含有一种转氨酶（TGase），它通过与海水或体液接触而被激活，这表明它可能通过蛋白质的交联来止血。为了阐明Tease的生理作用，对其胞内底物的鉴定和共同结构特性进行了研究。通过BrCN切割的15 k和12 k片段的氨基端序列YVAIQVLSL和GQKDSYVGDE，证实扇贝血细胞胞内底物45 kDa蛋白的主要成分是肌动蛋白。MDC是该酶的荧光底物，MDC特异性地掺入到肌球蛋白重链的S2区。B PLC分析BrCN-切割和蛋白酶消化的MDC掺入片段表明，Gln（520）是肌球蛋白重链上的第一个交联位点。Gln（520）也被鉴定为阿拉斯加青鳕肌球蛋白的主要mss连接位点。为了比较扇贝肌球蛋白重链与鱼类肌球蛋白重链的反应活性，首次通过cDNA序列分析测定了扇贝肌球蛋白重链的全氨基酸序列。扇贝肌球蛋白的交联位点位于含有Gln（520）的杆状区域的几个Gln残基上，表明底物蛋白之间的反应性存在差异，鱿鱼鳃TGas的细胞内底物分别为日本鱿鱼的不溶性190 kDa和90 kDa蛋白，枪乌贼的190 kDa组分。虽然两个190 kDa蛋白质的BrCN切割片段的氨基末端序列被确定为AIDVE和ANTDEAL，分别，没有显着的同源性，已知的蛋白质被检测到。由于200 kDa蛋白也被认为是扇贝血细胞TGase的主要胞内底物，因此这些蛋白被认为是海洋软体动物TGas的常见胞内底物。