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

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...

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Bibliographic Details
Published in:The Journal of biological chemistry 1985-09, Vol.260 (21), p.11567-11573
Main Authors: Kaunitz, J D, Gunther, R D, Sachs, G
Format: Article
Language:English
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
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Summary: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.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(17)39069-5