Characterization of the ATP-dependent proton pump of clathrin-coated vesicles

The ATP-dependent proton pump which was previously identified in clathrin-coated vesicles isolated from calf brain (Forgac, M., Cantley, L., Wiedenmann, B., Altstiel, L., and Branton, D. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 1300-1303) is further characterized. 7-Chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) was identified as a potent inhibitor of both ATP-dependent proton uptake and Mg2+-ATPase activity of coated vesicles. Thus, incubation with 10 microM NBD-Cl for 10 min at 23 degrees caused the loss of 80% of the Mg2+-ATPase activity and 95% of the proton pumping activity. The observed protection from NBD-Cl inhibition by ATP suggests that NBD-Cl may react at the catalytic site, and reversal of NBD-Cl inhibition by 2-mercaptoethanol is consistent with reaction at either a tyrosine or cysteine residue. In addition, no stable phosphorylated intermediate was observed during turnover of the coated vesicle proton pump and neither Na+ nor K+ was countertransported by the pump during ATP-dependent proton uptake.