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Study of mechanics

力学研究

基本信息

批准号：
12680629
负责人：
YOKOYAMA Ken
金额：
$ 2.3万
依托单位：
kanazawa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12680629/
关键词：
Thermus V-ATPase T.thermophilus ATP synthase

项目摘要

V-type ATPase (V_0V_1) capable of ATP-driven H^+ pumping and of H^+ gradient driven ATP synthesis was isolated from a thermophilic eubacterium, Thermus ther-mophilus. When the enzyme was analyzed by gel electrophoresis in the presence of sodium dodecyl sul-fate, it showed eight polypeptide bands of which four were subunits of V_1. We also isolated the V_0V_1 operon, containing nine genes in the order of atpG-I-L-E-X-F-A-B-D, which encoded proteins with molecular sizes of 13, 43, 10, 20, 35, 11, 64, 53, and 25 kDa, respectively. The last four genes were identified as those for V_0 subunits ; atpA, B, D, and F encoded the A, B, γ, and δ subunits, respectively. The first five genes, atpG-atpX, were identified as genes for the V_0 subunits. The product of atpL, the proteohpid subunit, lacked a 19-amino acid presequence and, unlike V-type ATPases, contained two membrane-spanning domains rather than four. The hydrophobic 43-kDa product of atpl is the smallest member so far found of the eukaryotic 100-kDa subunit family. Its electrophoretic band overlapped with the band of the A subunit. Therefore, all the gene products were found in our purified V_0V_1. We isolated the A_3B_3 subcomplex reconstituted from the isolated subunits and the A_3B_3γ subcomplex from subunit-expressing Escherichia coli. Electron microscopic observation of these subcomplexes revealed that the γ subunit of V_1 filled the central cavity of A_3B_3 and might be central subunit, similar to the γ subunit of F_1ATPase.

从嗜热真细菌Thermus thermophilus中分离到一种V型ATP酶（V_0V_1），该酶具有ATP驱动H^+泵送和H^+梯度驱动ATP合成的功能。在十二烷基硫酸钠存在下，用凝胶电泳分析该酶，显示八条多肽带，其中四条为V_1亚基。我们还分离了V_0V_1操纵子，其包含9个基因，顺序为atpG-I-L-E-X-F-A-B-D，其编码的蛋白质的分子大小分别为13、43、10、20、35、11、64、53和25 kDa。最后4个基因被鉴定为V_0亚基的基因; atpA、B、D和F分别编码A、B、γ和δ亚基。前5个基因atpG-atpX被鉴定为V_0亚基的基因。atpL的产物，proteohpid亚基，缺乏一个19个氨基酸的前序列，不像V型ATP酶，包含两个跨膜结构域，而不是四个。atpl的疏水性43-kDa产物是迄今为止发现的真核生物100-kDa亚基家族中最小的成员。其电泳带与A亚基的电泳带重叠。因此，在我们纯化的V_0V_1中发现了所有的基因产物。我们从分离的亚基和表达亚基的大肠杆菌中分离A_3B_3γ亚复合物。电镜观察表明，V_1的γ亚基填充在A_3B_3的中心空腔中，可能是中心亚基，类似于F_1 ATP酶的γ亚基。