Joint Study on the Mechanism of Insulin Action

在这项研究中，我们对PP 2C分子的结构域结构的阐明和对乳糜肌醇聚糖（CIG）的作用进行了生物化学研究，CIG已被弗吉尼亚大学的Joseph Larner博士和其他小组提出为胰岛素介体，对PP 2C同工型和衍生自PP 2C β的突变分子的酶活性的影响。1.构建了PP 2C β-1野生型、缺失突变体和点突变体的表达质粒。我们从大肠杆菌细胞提取物中纯化重组蛋白，并测定蛋白磷酸酶活性。从羧基末端缺失多达78个氨基酸不影响酶活性，而缺失100个氨基酸则完全消除了活性。另一方面，从氨基末端缺失11个氨基酸导致97%的酶活性损失，并且进一步缺失导致活性完全损失。替代任何t ...更多信息 在本研究中所测试的16种氨基酸中的6种特定氨基酸（E38、E60、H62、R179、R200和E243）导致酶活性损失98-100%。这些结果表明，PP 2C β至少由两个不同的功能结构域组成，一个氨基端的催化结构域约310个氨基酸，其余的羧基端结构域参与底物特异性的决定。已经确定，PP 2C活性的标准测定需要高浓度的Mg ^2+（Mg ^2+的Ka为1 mM）。然而，当CIG同时存在于检测混合物中时，即使Mg^<2+>浓度低至50 μ M，也能获得完全活性。用所有六种PP 2C亚型观察到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Mg^2+依赖性蛋白磷酸酶活性。这些结果表明，CIG是PP 2C异构体的激活剂，其作用点在PP 2C分子的催化结构域。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