Pedersen P L, Williams N, Hullihen J
Laboratory for Molecular and Cellular Bioenergetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Biochemistry. 1987 Dec 29;26(26):8631-7. doi: 10.1021/bi00400a021.
The ATPase activity of the F1 moiety of rat liver ATP synthase is inactivated when incubated prior to assay at 25 degrees C in the presence of MgCl2. The concentration of MgCl2 (130 microM) required to induce half-maximal inactivation is over 30 times higher than the apparent Km (MgCl2) during catalysis. Moreover, the relative efficacy of divalent cations in inducing inactivation during prior incubation follows an order significantly different from that promoting catalysis. Inactivation of F1-ATPase activity by Mg2+ is accompanied by the dramatic dissociation from the F1 complex of alpha subunits and part of the gamma-subunit population. The latter form a precipitate while the beta, delta, and epsilon subunits, and the remaining part of the gamma-subunit population, remain soluble. Dissociation is not a sudden "all or none" event but parallels loss of ATPase activity until alpha subunits have almost completely dissociated together with about 50% of the gamma-subunit population. Mg2+-induced loss of F1-ATPase activity cannot be prevented by including either the hydrolytic substrates ATP, GTP, or ITP in the incubation medium or the product ADP. Ethylenediaminetetraacetic acid, mercaptoethanol, and dithiothreitol are also ineffective in preventing loss of ATPase activity. Significantly, KPi at high concentration (greater than or equal to 200 mM) is effective in partially protecting F1 against inactivation. However, the most effective means of preventing Mg2+-induced inactivation of F1-ATPase activity is to rebind F1 to its F0 moiety in F1-depleted particles. When bound to F0, F1 is protected completely against divalent cation induced inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)
大鼠肝脏ATP合酶F1部分的ATP酶活性在检测前于25℃、MgCl2存在的条件下孵育时会失活。诱导半数最大失活所需的MgCl2浓度（130微摩尔）比催化过程中的表观Km（MgCl2）高30倍以上。此外，在预先孵育期间二价阳离子诱导失活的相对效力顺序与促进催化的顺序显著不同。Mg2+导致F1 - ATP酶活性失活的同时，α亚基和部分γ亚基群体从F1复合物中剧烈解离。后者形成沉淀，而β、δ和ε亚基以及γ亚基群体的其余部分保持可溶。解离不是突然的“全或无”事件，而是与ATP酶活性的丧失平行，直到α亚基几乎完全解离以及约50%的γ亚基群体解离。在孵育介质中加入水解底物ATP、GTP或ITP或产物ADP都无法阻止Mg2+诱导的F1 - ATP酶活性丧失。乙二胺四乙酸、巯基乙醇和二硫苏糖醇在防止ATP酶活性丧失方面也无效。值得注意的是，高浓度（大于或等于200毫摩尔）的KPi可有效部分保护F1不被失活。然而，防止Mg2+诱导F1 - ATP酶活性失活的最有效方法是将F1重新结合到F1耗尽颗粒中的F0部分。当与F0结合时，F1完全受到保护，不会被二价阳离子诱导失活。（摘要截短至250字）
