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.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000378:metabolism*; D017301:Ca(2+) Mg(2+)-ATPase; D002250:Carbon Radioisotopes; D002417:Cattle; D002966:Clathrin / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008744:Methylamines / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011188:Potassium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism
D002250:Carbon Radioisotopes; D002966:Clathrin; D008744:Methylamines; D010761:Phosphorus Radioisotopes; D000255:Adenosine Triphosphate; D012964:Sodium; C027451:methylamine; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium