DEACTIVATION OF F0F1 ATPASE IN INTACT PLANT-MITOCHONDRIA - EFFECT OF PH AND INHIBITORS

By using a method especially adapted to intact (pea leaf) mitochondria , we studied the regulation of the F0F1 ATPase by the electrochemical proton gradient (Delta mu(H)(+)) and by the matricial pH. The kinetics of decay of the ATP hydrolase activity was studied immediately after the collapse of the electrochemical proton gradient by an uncoupler. A t pH 7.5, three inhibitors of the ATPase (venturicidin, tri-n-butyl ti n and aurovertin), used at non-saturating concentrations, inhibited AT P hydrolysis to the same extent throughout the decay. This showed that the activity was totally controlled by the ATPase during all the deca y and rules out any involvement of the phosphate or nucleotide carrier s. This interpretation was confirmed by the fact that carboxyatractylo side, an inhibitor of the ATP/ADP antiporter, had a strong effect only on the initial rate of ATP hydrolysis, but not on the rate measured a fter some tens of seconds of decay. Oligomycin, at variance with the o ther ATPase inhibitors, interfered with the deactivation process, sugg esting that its effect depends on the conformational state of the enzy me. Between pH 6.5 and 7.5, the hydrolase activity rose continuously a nd was still kinetically controlled by the ATPase. At higher pH value, the activity slightly decreased and appeared limited by at least one of the carriers. The activity of the ATPase itself, free of any transp ort process, seemed to increase monotonously with pH from 6.5 to 8. Th e electrochemical proton gradient is required to maintain the ATPase a ctive, whereas no effect can be observed on transport processes. Matri cial pH, while modulating the apparent catalytic turnover, has no mark ed effect on the rate of deactivation. These results, obtained with in tact mitochondria, extend previous observations on the isolated enzyme and question the binding of IF1 as a rate-limiting step for ATPase de activation.