Kinetics of the release of the mitochondrial inhibitor protein. Correlation with synthesis and hydrolysis of ATP.
(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.
D000255:Adenosine Triphosphate / Q000096:biosynthesis / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D004789:Enzyme Activation; D006868:Hydrolysis; D007700:Kinetics; D008929:Mitochondria, Heart / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000097795:ATPase Inhibitory Protein
D000255:Adenosine Triphosphate; D011506:Proteins; D006180:Proton-Translocating ATPases; D000097795:ATPase Inhibitory Protein