Deactivation of F0F1 ATPase in intact plant mitochondria

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Abstract
By using a method especially adapted to intact (pea leaf) mitochondria, we studied the regulation of the F0F1 ATPase by the electrochemical proton gradient (Δμ˜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. At pH 7.5, three inhibitors of the ATPase (venturicidin, tri‐n‐butyl tin and aurovertin), used at non‐saturating concentrations, inhibited ATP hydrolysis to the same extent throughout the decay. This showed that the activity was totally controlled by the ATPase during all the decay and rules out any involvement of the phosphate or nucleotide carriers. This interpretation was confirmed by the fact that carboxyatractyloside, 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 after some tens of seconds of decay. Oligomycin, at variance with the other ATPase inhibitors, interfered with the deactivation process, suggesting that its effect depends on the conformational state of the enzyme. Between pH 6.5 and 7.5, the hydrolase activity rose continuously and 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 transport process, seemed to increase monotonously with pH from 6.5 to 8. The electrochemical proton gradient is required to maintain the ATPase active, whereas no effect can be observed on transport processes. Matricial pH, while modulating the apparent catalytic turnover, has no marked effect on the rate of deactivation. These results, obtained with intact mitochondria, extend previous observations on the isolated enzyme and question the binding of IF1 as a rate‐limiting step for ATPase deactivation.

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