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Joint Study on the Mechanism of Insulin Action

胰岛素作用机制的联合研究

基本信息

批准号：
07044218
负责人：
TAMURA Shinri
金额：
$ 4.61万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07044218/
关键词：
insulin action insulin mediator chyloinositol glycan protein phosphatase domain structure enzyme activity deletion mutant point mutant イノシトールプロテオグリカンゲノムDNA プロモーター領域

项目摘要

In this study we performed a biochemical study on the elucidation of the domain structure of PP2C molecule and on the effect of chyloinositol glycan (CIG), which has been proposed to be an insulin mediator by Dr.Joseph Larner of University of Virginia and other groups, on the enzyme activities of the PP2C isoforms and the mutant molecules derived from PP2Cbeta-1.We constructed the plasmids of the wild type of PP2C isoforms and the various deletion mutants and point mutants of PP2Cbeta-1 for expression in E.coli cells. We purified the rocombinant proteins from the E.coli cell extracts and determined the protein phosphatase activities. Deletion of up to 78 amino acids from the carboxy-terminal end did not affect the enzyme activity, whereas deletion of 100 amino acids totally eliminated the activity. On the other hand, deletion of 11 amino acids from the amino-terminal end caused a 97% loss of enzyme activity and further deletions caused a total loss of activity. Substitution of any of t … More he 6 specific amino acids (E38, E60, H62, R179, R200 and E243) among the 16 tested in this study caused 98-100% loss of enzyme activity. These observations suggested that PP2Cbeta is composed of at least two distinct functional domains, an amino-terminal catalytic domain of about 310 amino acids and the remaining carboxy-terminal domain, which is involved in determination of substrate specificity.Then, we determined the effect of CIG on the enzyme activities of wild type PP2C isoforms and the deletion mutants and point mutants. It is has been established that a high concentration (Ka for Mg^<2+>,1mM) of Mg^<2+> is required for the standard assay of PP2C activity. However, full activity was obtained even if the Mg^<2+> concentration was as low as 50muM when CIG was co-present in the assay mixture. This stimulatory effct of CIG was observed with all the six PP2C isoforms. The presence of CIG stimulated the activities of the deletion mutants and the point mutants as far as they had Mg^<2+> -dependent protein phosphatase activities. However, the mutants, which did no have Mg^<2+> -dependent activity, did not show any activity even in the presence of CIG.These results suggest that CIG acts as an activator of PP2C isoforms and that the point of action of CIG is in the catalytic domain of PP2C molecule. Less

在本研究中，我们对PP2C分子的结构域结构的阐明以及由弗吉尼亚大学的Joseph Larner博士等人提出的作为胰岛素介导物的乳清蛋白多糖(CIG)对PP2C异构体和PP2Cbeta-1突变分子的酶活性的影响进行了生化研究。我们从大肠杆菌细胞提取液中纯化了重组蛋白，并测定了其蛋白磷酸酶活性。羧基末端最多78个氨基酸的缺失不影响酶的活性，而100个氨基酸的缺失则完全消除了该酶的活性。另一方面，氨基端11个氨基酸的缺失会导致97%的酶活性损失，进一步的缺失会导致酶活性的完全丧失。替换t…中的任何一个在本研究的16个氨基酸中，有6个氨基酸(E38、E60、H62、R179、R200和E243)引起98-100%的酶活性损失。这些观察表明，PP2Cβ至少由两个不同的功能结构域组成，一个由大约310个氨基酸组成的氨基末端催化结构域和剩余的参与底物特异性决定的羧基末端结构域。然后，我们测定了CIG对野生型PP2C亚型以及缺失突变体和点突变体的酶活性的影响。已经确定，标准的PP2C活性测定需要较高浓度的镁离子(Ka为1 mM)。然而，当CIG存在于测定混合物中时，即使镁离子浓度低至50微米，仍可获得全部活性。用六种PP2C亚型观察了CIG的这种刺激作用。CIG的存在刺激了缺失突变体和点突变体的活性，只要它们具有依赖于镁的蛋白磷酸酶活性。然而，突变体不具有依赖于镁离子的活性，即使在CIG存在的情况下也没有表现出任何活性。这些结果表明，CIG是PP2C亚型的激活剂，CIG的作用点位于PP2C分子的催化区域。较少