Lippe G, Sorgato M C, Harris D A
Institute of Biochemistry, University of Padova, Italy.
Biochim Biophys Acta. 1988 Mar 30;933(1):1-11. doi: 10.1016/0005-2728(88)90050-3.
(1) The kinetics of the release of the mitochondrial inhibitor protein (IF1) is studied in bovine heart submitochondrial vesicles supplemented with 125I-labelled IF1, using a method for rapidly 'freezing' the state of F1-IF1 interaction. It is shown that generation of a protonmotive force leads to release of IF1 from F1 into solution, following an exponential process. (2) In one set of experiments the rate of IF1 release, in IF1 supplemented vesicles generating a protonmotive force, is correlated with the induction of ATP hydrolytic capacity. It is found that, even under different metabolic states (phosphorylating and non-phosphorylating conditions), both processes follow the same time-course (half-time of around 40 s) and that there is a direct correlation between induced ATPase capacity and IF1 released. This finding rules out the possibility of a non-inhibitory binding site for IF1 on the membrane. (3) In a second set of experiments, also using IF1 supplemented vesicles, the induction of the ATP hydrolytic capacity after energisation is correlated with the induction of the ATP synthetic capacity. Initial rates of both processes are monitored using firefly luciferase, keeping the assay systems as similar as possible. It is shown that the induction of each capacity follows an exponential time-course, with a half-time of around 40 s. This is in good agreement with the half-times obtained for the induction of ATP hydrolytic capacity and the rate of IF1 release, using the quench-stop method. (4) If the induction of ATP hydrolytic and synthetic capacities is followed in untreated submitochondrial vesicles, i.e., vesicles not supplemented with IF1, the extent and time-course of the change in both hydrolytic and synthetic capacities remain correlated, but the half-time of the transient falls to around 10 s. It is suggested that the length of the transient, observed in IF1 supplemented vesicles, results from partial loss of coupling during repeated centrifugations. (5) These results demonstrate that energy-dependent release of IF1 from F1 into solution results in a concomitant increase in both ATP synthetic and hydrolytic capacities of the ATP synthase complex, and that the time-course of this process is sensitive to the degree of coupling of the vesicles.
（1）采用一种快速“冻结”F1 - IF1相互作用状态的方法，在补充了125I标记的IF1的牛心亚线粒体囊泡中研究线粒体抑制剂蛋白（IF1）的释放动力学。结果表明，质子动力势的产生会导致IF1从F1释放到溶液中，遵循指数过程。（2）在一组实验中，在产生质子动力势的补充了IF1的囊泡中，IF1的释放速率与ATP水解能力的诱导相关。发现即使在不同的代谢状态（磷酸化和非磷酸化条件）下，这两个过程都遵循相同的时间进程（半衰期约为40秒），并且诱导的ATP酶活性与释放的IF1之间存在直接相关性。这一发现排除了IF1在膜上存在非抑制性结合位点的可能性。（3）在第二组实验中，同样使用补充了IF1的囊泡，通电后ATP水解能力的诱导与ATP合成能力的诱导相关。使用萤火虫荧光素酶监测这两个过程的初始速率，使测定系统尽可能相似。结果表明，每种能力的诱导都遵循指数时间进程，半衰期约为40秒。这与使用淬灭终止法获得的ATP水解能力诱导的半衰期和IF1释放速率非常一致。（4）如果在未处理的亚线粒体囊泡（即未补充IF1的囊泡）中跟踪ATP水解和合成能力的诱导，水解和合成能力变化的程度和时间进程仍然相关，但瞬变的半衰期降至约10秒。有人认为，在补充了IF1的囊泡中观察到的瞬变长度是由于重复离心过程中偶联的部分丧失所致。（5）这些结果表明，IF1从F1到溶液中的能量依赖性释放导致ATP合酶复合物的ATP合成和水解能力同时增加，并且该过程的时间进程对囊泡的偶联程度敏感。
