The lysosomal H+ pump: 8-azido-ATP inhibition and the role of chloride in H+ transport.
Lysosomes (tritosomes) were purified from the livers of rats injected with Triton WR 1339. The lysosomes developed an Mg2+-ATP-dependent pH gradient as measured by Acridine orange accumulation. H+ transport was supported by chloride, but not sulfate, and was independent of the cation used. H+ transport and Mg2+-stimulated ATPase was inhibited by diethylstilbesterol (K0.5 = 2 microM). N-Ethylmaleimide inhibited H+ transport (K0.5 = 30 microM). At low concentrations of N-ethylmaleimide, ATP partially protected H+ transport from inhibition with N-ethylmaleimide. Photolysis with 8-azido-ATP inhibited H+ transport and Mg2+-stimulated ATPase activity. Under these same conditions, 8-azido-[alpha-32P]ATP reacted with a number of polypeptides of the intact lysosome and lysosomal membranes. Pump-dependent potentials were measured using the fluorescent potential-sensitive dye, DiSC3(5) (3,3'-dipropylthiocarbocyanine) and ATP-dependent potential generation was inhibited by diethylstilbesterol. Chloride, but not sulfate reduced the magnitude of the ATP-dependent membrane potential, as measured using merocyanine 540. The chloride conductance, independent of ATP, was of sufficient magnitude to generate a H+ gradient driven by external chloride in the presence of tetrachlorosalicylanilide. In Cl- free media, ATP-dependent H+ transport was restored to control levels by outwardly directed K+ gradients in the presence of valinomycin. The role of cell Cl- is to provide the necessary conductance for supporting lysosomal acidification by the electrogenic proton pump.
D000165:Acridine Orange; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D001386:Azides / Q000494:pharmacology*; D001693:Biological Transport, Active; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000494:pharmacology; D051381:Rats; D013501:Surface-Active Agents / Q000494:pharmacology
D001386:Azides; D013501:Surface-Active Agents; D011092:Polyethylene Glycols; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D000165:Acridine Orange; C016811:tyloxapol