Covalent modification of the renal Na+/H+ exchanger by N,N'-dicyclohexylcarbodiimide.
We studied the effect of the carboxyl group-specific reagent N,N'-dicyclohexylcarbodiimide on the Na+/H+ exchanger present in microvillus membrane vesicles isolated from rabbit renal cortices. Pretreatment of membrane vesicles with dicyclohexylcarbodiimide resulted in irreversible inhibition of Na+/H+ exchange which was not due to vesicle disruption or collapse of imposed pH gradients. Inhibition by dicyclohexylcarbodiimide followed pseudo-first-order kinetics, resulted primarily from a decrease in binding affinity for substrate, was pH-dependent in a manner consistent with reaction with carboxyl groups, and was greater than inhibition by hydrophilic carbodiimides. Substrates Na+ and Li+ and the competitive inhibitor amiloride protected against inhibition by dicyclohexylcarbodiimide in a pH-dependent fashion. Finally, we demonstrated amiloride-sensitive covalent binding of radiolabeled dicyclohexylcarbodiimide to a 100-kDa protein. In conclusion, a catalytically important carboxyl group is located in a relatively hydrophobic microenvironment at or near the external transport site of the renal Na+/H+ exchanger; and the transporter itself, or a subunit thereof, may be a 100-kDa protein.
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D002234:Carbodiimides / Q000494:pharmacology*; D002352:Carrier Proteins / Q000378:metabolism*; D004024:Dicyclohexylcarbodiimide / Q000378:metabolism / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007672:Kidney Cortex / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008871:Microvilli / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers
D002234:Carbodiimides; D002352:Carrier Proteins; D017923:Sodium-Hydrogen Exchangers; D004024:Dicyclohexylcarbodiimide; D000584:Amiloride; D012964:Sodium