The lysosomal H + pump: 8-azido-ATP inhibition and the role of chloride in H + transport

Abstract

Lysosomes (tritosomes) were purified from the livers of rats injected with Triton WR 1339. The lysosomes developed an Mg 2+-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 Mg 2+-stimulated ATPase was inhibited by diethylstilbesterol ( K 0.5 = 2 μM). N-Ethylmaleimide inhibited H + transport ( K 0.5 = 30 μM). 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 Mg 2+-stimulated ATPase activity. Under these same conditions, 8-azido-[α- 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, DiSC 3(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.