Covalent modification of the renal Na+/H+ exchanger by N,N'-dicyclohexylcarbodiimide

P. Igarashi, P. S. Aronson

Research output: Contribution to journalArticle

37 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)860-868
Number of pages9
JournalJournal of Biological Chemistry
Volume262
Issue number2
StatePublished - Jan 1 1987

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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