Inhibition byN-ethylmaleimide of the MgATP-driven proton pump of the chromaffin granules

Abstract

The thiol reagent N-ethylmaleimide (NEM) completely inhibits the proton pump activity of the H⁺-ATPase in chromaffin granule ‘ghosts’ at concentrations which only partly (∼20%) inhibit the Mg²⁺-dependent ATP hydrolysis. Half-maximal inhibition was obtained at ∼13 μM NEM as compared to 18 μM for the classical proton channel inhibitor N,N'-dicyclohexylcarbodiimide (DCCD), and the apparent stoichiometry of the inhibitors at complete inhibition was NEM:DCCD ⋍ 1 : 2. High concentrations of NEM (⪢100 μM) induce a dissipation of the transmembrane potential generated by MgATP. These findings establish NEM as a valuable proton channel inhibitor in chromaffin granules and explain the rather complex effect of NEM previously reported for catecholamine accumulation in this organelle.