Characterization of an electrogenic ATP and chloride-dependent proton translocating pump from rat renal medulla

• Published in: The Journal of biological chemistry Vol. 260; no. 21; pp. 11567 - 11573
• Main Authors: Kaunitz, J D, Gunther, R D, Sachs, G
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 25.09.1985; American Society for Biochemistry and Molecular Biology
• Subjects: Adenosine Triphosphatases - analysis; Adenosine Triphosphate - pharmacology; Animals; Biological and medical sciences; Biological Transport; Cell physiology; Chlorides - pharmacology; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; Kidney Medulla - metabolism; Kinetics; Male; Membrane and intracellular transports; Molecular and cellular biology; Nephrons - metabolism; Permeability; Protons; Rats; Rats, Inbred Strains; Active transport; Rat; Rodentia; Ion transport; Kidney; Characterization; Vertebrata; Mammalia; Membrane pump; Animal; Chlorides; Dependence; Proton; Renal medulla; ATP
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)39069-5

To study acidification mechanisms in the distal nephron, microsomes were prepared from rat renal medulla by differential centrifugation. Microsomes were enriched in the enzyme marker gamma-glutamyl transferase and contained an ATP-dependent proton pump, as evidenced by ATP-dependent, 3,3',4',5-tetrachlorosalicylanilide-reversible quenching of acridine orange fluorescence. Acidification was vanadate-insensitive, but was completely inhibited by micromolar N-ethylmaleimide. Maximal acidification was achieved in the presence of halide (Cl-, Br-) only and was not attainable with potassium-valinomycin diffusion potentials without halide ion. Microsomal ATPase activity was neither chloride- nor N-ethylmaleimide-sensitive. A chloride conductance was observed only with vesicles which had undergone ATP-dependent acidification. An ATP-dependent, N-ethylmaleimide-inhibitable, 3,3',4',5-tetrachlorosalicylanilide-reversible, and chloride-attenuated quench of bis(1,3-dibutylbarbituric acid-(5] pentamethinoxonol fluorescence was seen, consistent with net transfer of positive charge into the vesicles. Nonetheless, positive intravesicular potentials increased the ATP-dependent initial acidification rate, perhaps by increasing availability of chloride ion to the transport site. Our results are consistent with an electrogenic, ATP-dependent proton pump regulated by a voltage-sensitive chloride site.