Joint Study on the Mechanism of Insulin Action

在本研究中，我们对PP2C分子结构域的解析和乳酸菌肌醇聚糖（CIG）的作用进行了生化研究，乳酸菌肌醇聚糖（CIG）是由弗吉尼亚大学的joseph Larner博士等人提出的胰岛素介质，对PP2C异构体和pp2c2 -1衍生的突变分子的酶活性的影响。我们构建了野生型PP2C亚型和pp22c -1的各种缺失突变体和点突变体的质粒，用于在大肠杆菌细胞中表达。我们从大肠杆菌细胞提取物中纯化了重组蛋白，并测定了蛋白磷酸酶活性。从羧基末端删除多达78个氨基酸不影响酶的活性，而删除100个氨基酸完全消除了酶的活性。另一方面，从氨基末端缺失11个氨基酸导致酶活性损失97%，进一步缺失导致酶活性全部丧失。在本研究测试的16种氨基酸中，有6种特异性氨基酸（E38、E60、H62、R179、R200和E243）被取代，酶活性损失高达98-100%。这些观察结果表明，PP2Cbeta至少由两个不同的功能域组成，一个氨基端催化域约310个氨基酸，另一个羧基端参与底物特异性的确定。然后，我们测定了CIG对野生型PP2C异构体、缺失突变体和点突变体酶活性的影响。已经确定，PP2C活性的标准测定需要高浓度的Mg^<2+> （Ka为Mg^<2+>,1mM）。然而，当CIG同时存在于实验混合物中时，即使Mg^<2+>浓度低至50muM，也能获得充分的活性。CIG的这种刺激作用在所有6种PP2C亚型中都被观察到。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Mg^<2+>依赖的蛋白磷酸酶活性。然而，没有Mg^<2+>依赖性活性的突变体即使在CIG存在下也没有表现出任何活性。这些结果表明，CIG作为PP2C同工异构体的激活剂，CIG的作用点在PP2C分子的催化结构域中。少

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