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应用中最重要的一点是其极高的稳定性。人工膜的重构已经实现，并且有多种生物传感器投入实际应用。然而，这些制剂的稳定性并不令人满意。为此，研究主要集中在以下两种超嗜热酶上：a.超嗜热V型ATP酶的稳定性和功能研究，该酶的同源膜ATP酶已被报道。所用的超嗜热菌为脱硫球菌，并与嗜温菌进行比较。b.控制蛋白质高级结构的超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，因此克隆是基于该结构。使用的超嗜热菌是脱硫球菌和热球菌。a.超嗜热和嗜温V-ATP酶之间的稳定残基交换的鉴定基于计算机同源性搜索和X射线晶体学。最丰富的稳定残基交换是Asp到Glu（螺旋稳定），其次是Ser到Ala，Gly到Ala（增强内部疏水性）。b.在这些基因克隆后分离出包括伴侣蛋白和消旋酶的分子伴侣。这些分子伴侣与真细菌嗜热菌的 HSP70 有很大不同。对超嗜热分子伴侣的详细分析揭示了其αβ亚基（J.Mol.Biol.273：635，1997）。最后，这些研究为J.E.Walker和P.D.Boyer关于ATP酶的诺贝尔奖（1997）做出了贡献。 Walker 是我们关于嗜热 ATP 酶 X 射线晶体学分析报告的合著者。 Boyer 经常引用 Ann 中关于嗜热 ATP 酶的著作。 Rev.of Biochem.，并将于 1998 年 3 月在济池医学院给我们做一个关于这个主题的讲座。