Structure-function relationships of the Escherichia coli ATP synthase probed by trypsin digestion.
Structure-function relationships of the Escherichia coli ATP synthase probed by trypsin digestion.
复制标题
通过胰蛋白酶消化探测大肠杆菌 ATP 合酶的结构与功能关系。
DOI：
10.1021/bi00402a016
复制
发表时间：
1988
期刊：
影响因子：
2.9
通讯作者：
Capaldi,RA
中科院分区：
文献类型：
--
作者：
Gavilanes-Ruiz,M;Tommasino,M;Capaldi,RA
1987年9月21日摘要：胰蛋白酶切割已被用于探测大肠杆菌ATP合酶（ECFiF 0）的结构-功能关系。胰蛋白酶在分离的ECFI中切割所有五个亚基a、f3、y、δ和e。α亚基的切割涉及N-末端15个残基的去除，β亚基在C-末端附近被切割，γ亚基在Ser 202附近被切割，δ和ε亚基似乎在几个位点被切割以产生小的肽片段。胰蛋白酶切割ECFi增强ATP酶活性之间的6- 8倍，在不同的制剂，在一个时间过程中，随后的e亚基的切割。这种去除的e亚基增加多位点ATP酶活性，但不unplanted ATP酶活性，表明t亚基的抑制作用是由于协同效应。发现洗涤剂月桂基二甲基氧化胺可提高多位催化作用，也可使不饱和催化作用提高2倍以上。长时间的胰蛋白酶切割留下了一个高活性的ATP酶，它只含有α和β亚基以及两个γ亚基片段。在与分离的ECFi中相同的位点处，ECFi的所有亚基在ECFiFO制剂中被胰蛋白酶裂解。两个亚基，β和ε亚基，在ECFiF 0中以与单独的ECFi中相同的速率被切割。α、γ和δ亚基在ECFiFO中裂解显著更慢。F0部分的亚基B在ECFiF 0中仅缓慢裂解，但当加入去污剂月桂基二甲基氧化胺以破坏ECFi和α亚基之间的相互作用时，α、γ、δ和B亚基全部快速裂解。
Revised Manuscript Received September 21, 1987 abstract: Trypsin cleavage has been used to probe structure-function relationships of the Escherichia coli ATP synthase (ECFiF0). Trypsin cleaved all five subunits, a, f3, y, 8, and e, in isolated ECF!. Cleavage of the a subunit involved the removal of the N-terminal 15 residues, the (3 subunit was cleaved near the C-terminus, the y subunit was cleaved near Ser202, and the 8 and e subunits appeared to be cleaved at several sites to yield small peptide fragments. Trypsin cleavage of ECFi enhanced the ATPase activity between 6-and 8-fold in different preparations, in a time course that followed the cleavage of the e subunit. This removal of the e subunit increased multisite ATPase activity but not unisite ATPase activity, showing that the inhibitory role of the t subunit is due to an effect on cooperativity. The detergent lauryldimethylamine oxide was found to increase multisite catalysis andalso increase unisite catalysis more than 2-fold. Prolonged trypsin cleavage left a highly active ATPase containing only the a and (3 subunits along with two fragments of the y subunit. All of the subunits of ECFi were cleaved by trypsin in preparations of ECFiF0 at the same sites as in isolated ECFi. Twosubunits, the (3 and e subunits, were cleaved at the same rate in ECFiF0 as in ECFi alone. The a, y, and 5 subunits were cleaved significantly more slowly in ECFiF0. Subunit b of the F0 part was cleaved only slowly in ECFiF0, but the a, y, 8, and b subunits were all cleaved rapidly when the detergent lauryldimethylamine oxide was added to disrupt the interaction between ECFi and the