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

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构建了PP2C同工型的野生类型的质粒以及PP2CBETA-1的各种缺失突变体和点突变体，用于在大肠杆菌细胞中的表达。我们从大肠杆菌细胞提取物中纯化了罗科组蛋白，并确定了蛋白质磷酸酶活性。从羧基末端删除多达78个氨基酸不会影响酶活性，而100个氨基酸的删除完全消除了活性。另一方面，从氨基末端删除11个氨基酸，导致酶活性损失97％，进一步的缺失导致了总活性的总损失。在这项研究中测试的16个中，替代了任何一个t…更多的HE HE 6特异性氨基酸（E38，E60，H62，R179，R200和E243），导致酶活性损失98-100％。这些观察结果表明，PP2CBETA由至少两个不同的功能结构域组成，即约310个氨基酸的氨基末端催化结构域和其余的羧基末端结构域，其中涉及确定底物特异性。然后，我们确定了CIG对野生型PP2C同工型以及缺失突变体和点突变体的酶活性的影响。已经确定了Mg^<2+>的高浓度（Mg^<2+>，1mm）的高浓度是PP2C活性的标准测定所必需的。但是，即使Mg^<2+>浓度在测定混合物中共同呈现时，也获得了全部活性。使用所有六个PP2C同工型观察到这种刺激的CIG效果。 CIG的存在刺激了缺失突变体的活性和点突变体，并且它们具有mg^<2+>依赖性蛋白质磷酸酶活性。但是，没有具有mg^<2+>依赖性活性的突变体，即使在CIG存在的情况下也没有显示任何活性。这些结果表明CIG充当PP2C同工型的激活剂，而CIG的作用点在PP2C分子的催化结构域中。较少的