Interaction of anions and ATP with the coated vesicle proton pump.
ATP-driven proton transport in intact clathrin-coated vesicles requires the presence of a permeant anion, such as Cl-, to provide charge compensation during the electrogenic movement of protons. Using the purified (H+)-ATPase from clathrin-coated vesicles in both the detergent-solubilized and reconstituted states, we have studied the direct effects of anions on the activity of this enzyme. Both proton transport and ATP hydrolysis by the purified enzyme are independent of the presence of Cl-. In addition, proton transport does not occur even at high Cl- concentrations unless K+ and valinomycin are present to dissipate the membrane potential generated. These results indicate that the anion channel which provides for Cl- flux in intact coated vesicles is not a component of the purified (H+)-ATPase. Inhibition of ATPase activity is observed in the presence of I-, NO3-, or SO4(2-), with 50% inhibition occurring at 350 mM I-, 50 mM NO3-, or 40 mM SO4(2-). The presence of ATP lowers the concentration of I- required for 50% inhibition from 350 mM to 100 mM and increases the maximal inhibition observed in the presence of NO3- from 65% to 100%. Two separate mechanisms appear to be responsible for anion inhibition of the (H+)-ATPase. Thus, I- and high concentrations of NO3- (in the presence of ATP) cause inhibition by dissociation of the (H+)-ATPase complex, while SO4(2-) and NO3- (in the absence of ATP) cause inhibition without dissociation of the complex, suggesting the existence of an inhibitory anion binding site on the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
Arai
H
H
Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
Pink
S
S
Forgac
M
M
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D000838:Anions; D001921:Brain / Q000201:enzymology; D002417:Cattle; D002712:Chlorides / Q000494:pharmacology*; D003034:Coated Pits, Cell-Membrane / Q000201:enzymology*; D011992:Endosomes / Q000201:enzymology*; D007700:Kinetics; D006180:Proton-Translocating ATPases / Q000378:metabolism*
D000838:Anions; D002712:Chlorides; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases