Wu J H, Li S G, Lin Z H
Institute of Biophysics, Academia Sinica, Beijing, People's Republic of China.
Biochim Biophys Acta. 1994 Mar 29;1185(1):50-5. doi: 10.1016/0005-2728(94)90192-9.
Mutant strain AN1518 or AN2387 (Gly48-->Asp in epsilon-subunit) and partial revertant strain AN2540 (Gly48-->Asp, Pro47-->Ser in epsilon-subunit) of E. coli were used in a kinetic study of membrane-bound H(+)-ATPase. It was found that at pH 9.0 mutant strain AN1518 or AN2387 and partial revertant strain AN2540 gave a low initial rate, which increased with time until linearity was reached after 1-2 min. This phenomenon was prominent in mutant strains, but was not so obvious in wild-type AN346 of E. coli; this property is similar to F1-ATPase reported by Cox [1]. The mechanism of the slow activation of membrane-bound H(+)-ATPase was further investigated in this paper. The experimental results indicated that the hydrolytic rate of E. coli F1F0-ATPase that increased with time was membrane protein concentration- and pH-dependent, and that the product ADP produced during ATP hydrolysis is the factor causing the slow activation. Preincubation of the hydrolytic product ADP with a concentration comparable to that produced in the assay (20 microM) caused initial activation of ATP hydrolysis and abolished the slow activation. On the other hand, with the removal of ADP during the progress of the hydrolytic reaction it could be seen that the slow activation was abolished as well. In order to test the relationship between the epsilon-subunit and ADP involved in the slow activation, trypsin treatment was carried out on the membrane-bound H(+)-ATPase of various strains. The activation observed after trypsin treatment was on the order of AN1518 > AN2540 > AN346. The activation effects of ADP and trypsin were not found to be additive. This implies that ADP acted in a similar way to trypsin, i.e., to cause removal of the epsilon-subunit. A tentative mechanism of the slow activation was proposed that ADP, a product of ATP hydrolysis, could induce conformational changes of F1F0 at alkaline pH 9.0, thus weakening the binding strength between the epsilon-subunit and other subunits of F1F0, and resulting in removal or partial removal of the epsilon-subunit. This further impaired the coupling of F1 and F0 in the mutant strains; as a consequence the rate of ATP hydrolysis was increased.
大肠杆菌的突变株AN1518或AN2387（ε亚基中的Gly48→Asp）以及部分回复突变株AN2540（ε亚基中的Gly48→Asp，Pro47→Ser）被用于膜结合H(+)-ATP酶的动力学研究。发现在pH 9.0时，突变株AN1518或AN2387以及部分回复突变株AN2540的初始速率较低，该速率随时间增加，直至1 - 2分钟后达到线性。这种现象在突变株中很突出，但在大肠杆菌野生型AN346中不那么明显；这种特性与Cox [1]报道的F1-ATP酶相似。本文进一步研究了膜结合H(+)-ATP酶缓慢激活的机制。实验结果表明，随时间增加的大肠杆菌F1F0-ATP酶水解速率与膜蛋白浓度和pH有关，并且ATP水解过程中产生的产物ADP是导致缓慢激活的因素。将水解产物ADP以与测定中产生的浓度相当的浓度（20 microM）进行预孵育，会导致ATP水解的初始激活并消除缓慢激活。另一方面，在水解反应过程中去除ADP，可以看到缓慢激活也被消除。为了测试参与缓慢激活的ε亚基与ADP之间的关系，对各种菌株的膜结合H(+)-ATP酶进行了胰蛋白酶处理。胰蛋白酶处理后观察到的激活程度为AN1518 > AN2540 > AN346。未发现ADP和胰蛋白酶的激活作用具有加和性。这意味着ADP的作用方式与胰蛋白酶类似，即导致ε亚基的去除。提出了一种缓慢激活的初步机制，即ATP水解产物ADP在碱性pH 9.0时可诱导F1F0的构象变化，从而削弱ε亚基与F1F0其他亚基之间的结合强度，导致ε亚基的去除或部分去除。这进一步损害了突变株中F1和F0的偶联；结果ATP水解速率增加。
