Characteristics of the proton pump in rat renal cortical endocytotic vesicles.
The characteristics of the H+ pump in isolated rat renal endocytotic vesicles were studied by the delta pH-sensitive dye acridine orange, the voltage-sensitive dye 3,3'-dipropylthiadicarbocyanine iodide, and by a coupled optical ATPase assay. Intravesicular acidification depended on ATP and Mg2+ concentrations with half-maximal activations at 73 and 77 microM, respectively. CTP, GTP, UTP, and ITP partially supported acidification, but ADP and AMP did not. Ouabain, ethoxzolamide, levamisole, and vanadate did not inhibit H+ uptake into endocytotic vesicles. Oligomycin inhibited partially. Depending on concentration and preincubation time, Dio-9, filipin, N-ethylmaleimide (NEM), and dicyclohexylcarbodiimide (DCCD) inhibited H+ uptake completely. Filipin and, partially, DCCD acted nonspecifically by dissipating pH gradients. A specific cation was not required for the H+ pump; Zn2+ inhibited. Compared with mannitol, ATP-driven H+ uptake was stimulated by SCN- greater than Cl- greater than Br- greater than I- much greater than HPO4(2-) = gluconate = HCO3- = F-, but not by SO4(2-), NO3-, CH3COO-, S2O3(2-), and S4O6(2-). Chloride stimulated H+ uptake from the outside of the vesicles with an apparent Km of 27 mM. In the absence of Cl-, ATP-driven proton uptake was increased by intravesicular K+ and valinomycin, suggesting that the pump is electrogenic. The electrogenicity, however, could not be demonstrated with voltage-sensitive dyes. The vesicle membrane contains no significant K+ and Cl- conductances; only a conductance for H+ was found. The vesicles exhibited an ouabain-, oligomycin-, and vanadate-insensitive ATPase activity that was inhibited by DCCD and NEM. Our data indicate the presence of an electrogenic H+ pump in endocytotic vesicles from rat renal proximal tubules with similar characteristics as H+ pumps present in various intracellular (nonmitochondrial) membranes.
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Sabolić
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Burckhardt
D000818:Animals; D000838:Anions; D002412:Cations; D002712:Chlorides / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology; D004553:Electric Conductivity; D004563:Electrochemistry; D004705:Endocytosis; D006859:Hydrogen / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007473:Ion Channels / Q000502:physiology*; D007672:Kidney Cortex / Q000201:enzymology / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D009711:Nucleotides / Q000502:physiology; D009994:Osmolar Concentration; D011188:Potassium / Q000378:metabolism / Q000502:physiology; D006180:Proton-Translocating ATPases / Q000378:metabolism; D011522:Protons; D051381:Rats
D000838:Anions; D002412:Cations; D002712:Chlorides; D007473:Ion Channels; D009711:Nucleotides; D011522:Protons; D006859:Hydrogen; D006180:Proton-Translocating ATPases; D008274:Magnesium; D011188:Potassium