INTERACTION OF THE CLATHRIN-COATED VESICLE V-ATPASE WITH ADP AND SODIUM-AZIDE

The kinetics of adenosine triphosphate (ATP)-dependent proton transpor t into clathrin-coated vesicles from bovine brain have been studied. W e observe that the vacuolar proton-translocating ATPase (V-ATPase) fro m clathrin-coated vesicles is subject to two different types of inhibi tion by ADP. The first is competitive inhibition with respect to ATP, with a K-i for ADP of 11 mu M. The second type of inhibition occurs af ter preincubation of the V-ATPase in the presence of ADP and Mg2+, whi ch results in inhibition of the initial rate of proton transport follo wed by reactivation over the course of several minutes. The second eff ect is observed at ADP concentrations as low as 0.1-0.2 mu M, indicati ng that a high affinity inhibitory complex is formed between ADP and t he V-ATPase and is only slowly dissociated after the addition of ATP. We have further investigated the effect of sodium azide, an inhibitor of the F-ATPases that has been shown to stabilize an inactive complex between ADP and the F-1-F-0-ATP synthase (F-ATPase), We observed that azide inhibited ATP-dependent proton transport by the purified, recons tituted V-ATPase with a K-0.5 of 0.2-0.4 mw but had no effect on ATP h ydrolysis. Azide was shown not to increase the passive proton permeabi lity of reconstituted vesicles and did not stimulate ATP hydrolysis by the reconstituted enzyme, in contrast with CCCP, which both abolished the proton gradient and stimulated hydrolysis, Thus, azide does not a ppear to act as a simple uncoupler of proton transport and ATP hydroly sis. Rather, azide may have some more direct effect on V-ATPase activi ty. Possible mechanisms by which azide could exert this effect on the V-ATPase and the contrasting effects of azide on the F- and V-ATPases are discussed.