title: The lysosomal proton pump is electrogenic.
abstract: Lysosomes were purified approximately 40-fold from rat kidney cortex by differential and Percoll density gradient centrifugation. In a sucrose medium, the lysosomes quenched the fluorescence of the potential sensitive dye diS-C3-(5) (3,3'-dipropylthiocarbo-cyanine iodide) in a time-dependent manner, indicating that the dye accumulates within the lysosomal interior. After treatment of the lysosomes with valinomycin, the dye fluorescence displayed a logarithmic dependence upon the external K+ concentration; thus, the fluorescence signal provides a semiquantitative measure of the lysosomal membrane potential (delta psi). In the absence of valinomycin, lysosomal quenching of diS-C3-(5) fluorescence was partially reversed by agents which collapse the lysosomal pH gradient (ammonium sulfate, chloroquine, and K nigericin), suggesting that the proton gradient across the lysosomal membrane contributes to delta psi. A rapid increase in diS-C3-(5) fluorescence, indicative of an increase in delta psi, was observed upon the addition of Mg-ATP to the lysosomes. The ATP-dependent fluorescence change was inhibited by protonophores, K valinomycin, permeable anions, and N-ethylmaleimide, but was unaffected by ammonium sulfate, K nigericin, or sodium vanadate. Oligomycin had no effect at concentrations below 2 micrograms/ml; at higher concentrations, oligomycin partially inhibited the fluorescence response to Mg-ATP, but it also inhibited the fluorescence response to K valinomycin, suggesting that it had modified the permeability of the lysosomal membrane. Dicylohexylcarbodiimide behaved similarly to oligomycin. Mg-ATP also altered the lysosomal distribution of 86Rb+ (in the presence of valinomycin) and S[14C]CN-, consistent with an increase in the potential of the lysosomal interior of 40-50 mV. The results demonstrate that the lysosomal proton pump is electrogenic.
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002458:Cell Fractionation; D004594:Electrophysiology; D005033:Ethylmaleimide / Q000494:pharmacology; D007672:Kidney Cortex / Q000648:ultrastructure; D008247:Lysosomes / Q000201:enzymology*; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D006180:Proton-Translocating ATPases; D051381:Rats; D014634:Valinomycin / Q000494:pharmacology
chemical_list: D014634:Valinomycin; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases; D005033:Ethylmaleimide