Study of mechanics

从嗜热真细菌Thermus thermophilus中分离到一种具有ATP驱动的H~+泵和H~+梯度驱动的ATP合成的V型ATPase(V0V1)。在十二烷基硫酸钠存在下，该酶经凝胶电泳法分析，显示出8条多肽带，其中4条是V_1的亚基。我们还分离到V_0V_1操纵子，包含9个基因，其顺序为atpG-I-L-E-X-F-A-B-D，分别编码13、43、10、20、35、11、、53和25 kDa的蛋白质。后4个基因分别编码A、B、γ和δ亚基。前5个基因atpG-atpX被鉴定为V0亚基基因。与V型ATPase不同的是，ATPL的产物缺乏19个氨基酸的前序列，并且包含两个跨膜结构域，而不是四个。ATPL的43 kDa疏水产物是迄今为止发现的真核100 kDa亚单位家族中最小的成员。其电泳带与A亚基的带重叠。因此，在我们纯化的V_0V_1中发现了所有的基因产物。我们从分离的亚基中分离到了重组的A_3B_3亚基，并从表达亚基的大肠杆菌中分离到了A_3B_3的γ亚复合体。这些亚复合体的电子显微镜观察表明，V_1的γ亚基填充在A_3B_3的中央空腔中，可能是与F_1ATPase的γ亚基相似的中央亚基。

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.