Biochemical aspects of H(+)-ATPase in renal proximal tubules: inhibition by N,N'-dicyclohexylcarbodiimide, N-ethylmaleimide, and bafilomycin.
Brush-border membranes from rat kidney cortex are transiently exposed to cholate to reorient ATP-driven H+ pumps to the outside of the vesicles. The carboxyl group reagent, N,N'-dicyclohexylcarbodiimide (DCCD), inhibits ATP-driven H+ uptake into cholate-pretreated vesicles irreversibly. Complete inhibition requires treatment of vesicles with 0.2 mM DCCD for greater than or equal to 15 min. ATP and ADP do not protect the H+ pump from inactivation suggesting that DCCD modifies pump subunits involved in H+ translocation, but not those related to ATP hydrolysis. With [14C]DCCD a 16 kDa protein is strongly labeled in brush-border and endosomal membranes, but not in basolateral membranes. Molecular mass of this protein and distribution similar to H(+)-ATPases suggest a role as H(+)-conducting subunit of the H+ pumps. The SH-group reagent, N-ethylmaleimide (NEM), also inhibits ATP-driven H+ uptake irreversibly. As opposed to DCCD, ATP and ADP protect the pump from irreversible inhibition indicating that NEM modifies SH-groups in the proximity of ATP hydrolysis sites. Finally, 15 nM of a potent inhibitor of vacuolar ATPases, bafilomycin B1, abolishes ATP-driven H+ uptake. Inactivation by DCCD and NEM, labeling of 16 kDa subunits by [14C]DCCD, and high sensitivity to bafilomycin indicate that the H+ pump (H(+)-ATPase) in rat renal brush-border membranes belongs to the class of vacuolar ATPases. Bafilomycin may prove a valuable tool for specific inhibition of the renal H(+)-ATPase in future studies.
Jehmlich
K
K
Max-Planck-Institut für Biophysik, Frankfurt/Main, Federal Republic of Germany.
Sablotni
J
J
Simon
B J
BJ
Burckhardt
G
G
D000255:Adenosine Triphosphate / Q000502:physiology; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001693:Biological Transport, Active / Q000187:drug effects; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D005033:Ethylmaleimide / Q000494:pharmacology*; D006859:Hydrogen / Q000494:pharmacology; D007687:Kidney Tubules, Proximal / Q000201:enzymology*; D018942:Macrolides; D011506:Proteins / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D051381:Rats
D000900:Anti-Bacterial Agents; D018942:Macrolides; D011506:Proteins; D004024:Dicyclohexylcarbodiimide; D006859:Hydrogen; C052501:bafilomycin B1; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D005033:Ethylmaleimide