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Hyperthermophilic enzymes suitable for functioning membranes.

适用于功能膜的超嗜热酶。

基本信息

批准号：
07558128
负责人：
KAGAWA Yasuo
金额：
$ 6.02万
依托单位：
Jichi Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07558128/
关键词：
hyperthermphile chaperone biomembrane HSP70 racemase ATPase reconstitution heat shock HSP70 Hyperthermophile V-type ATPase

项目摘要

The most important point in the application of hyperthermophilic enzymes for an artificial functional membrane is their extreme stability. The reconstitution of the artificial membrane has already been achieved, and there are many biosensors in practical uses. However, the stability of these preparations is not satisfactory.For this reason, the research was focus on the following two hyperthermophilic enzymes :a.The stability and functional study on the hyperthermophilic V-type ATPase, for which homological membrane ATPases have already been reported. The hyperthermophile used was Desulfurococcus which was compared with mesophiles.b.The isolation and functional study on the hyperthermphilic chaperones which controls the higher structure of proteins.Since both membrane ATPases and chaperones contain common ATPase structure, cloning was based on the structure. The hyperthermophiles used were Desulfurococcus and Thermococcus.a.The identification of the stabilizing residue exchanges between the hyperthermophilic and mesophilic V-ATPases was based on the computerized homology search and the X-ray crystallography. The most abundant stabilizing residue exchange was Asp to Glu (helixstabilizing), and the next were Ser to Ala, and Gly to Ala (enhancing the internal hydrophobicity).b.The chaperones including chaperonin and racemase was isolated after the cloning of these genes. The chaperones were quite different from HSP70 of the eubacterial thermophiles. The detailed analysis of the hyperthermophilic chaperone revealed its alpha beta subunits (J.Mol.Biol.273 : 635,1997).Finally, these studies contributed to Nobel Prize (1997) won by J.E.Walker and P.D.Boyer on the ATPase. Walker is the coauthor of our report on X-ray crystallographic analysis of thermophilic ATPase. Boyer frequently quoted works on thermophilic ATPases in Ann. Rev.of Biochem., and will give us a lecture on this subject in Jichi Medical School in March 1998.

超嗜热酶在人工功能膜中的应用最重要的一点是它们的极端稳定性。人工膜的重构已经实现，并且有许多生物传感器在实际应用中。因此，本研究主要集中在以下两种超嗜热酶：a.超嗜热V型ATP酶的稳定性和功能研究，其同源膜ATP酶已有报道。使用的嗜热菌是Desulfurococcus，并与嗜温菌进行了比较。B.控制蛋白质高级结构的嗜热分子伴侣的分离和功能研究。由于膜ATP酶和分子伴侣都含有共同的ATP酶结构，因此克隆是基于结构的。所用的超嗜热菌是Desulfurococcus和Thermococcus。a.超嗜热和中温V-ATP酶之间稳定残基交换的鉴定是基于计算机同源搜索和X射线晶体学。其中以天冬氨酸与谷氨酸的交换最多（螺旋稳定），其次是丝氨酸与丙氨酸的交换，以及甘氨酸与丙氨酸的交换（增强了内疏水性）。B.克隆了这些基因后，分离得到了分子伴侣蛋白和消旋酶。这些分子伴侣与真细菌嗜热菌的HSP 70有很大的不同。对超嗜热分子伴侣蛋白的详细分析揭示了其α β亚基（J.Mol.Biol.273：635，1997），并为步行者和Boyer获得1997年诺贝尔奖做出了贡献。步行者是我们关于嗜热ATP酶的X射线晶体学分析报告的合著者。Boyer经常引用Ann. Rev.of Biochem.中关于嗜热ATP酶的著作，并将于1998年3月在自治医科大学为我们做一次关于这个主题的讲座。