Inhibition by N‐ethylmaleimide of the MgATP‐driven proton pump of the chromaffin granules

[摘要]

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.

[学科分类] 生物化学/生物物理

[关键词] Proton pump;Proton channel;H+-ATPase;Chromaffin granules;N-ethylmaleimide;Adrenal medulla;ANS;1-anilinonaphthalene 8-sulphonic acid;BES;N;N-bis(2-hydroxyethyl)-2-amino-ethanesulfonic acid;DCCD;N;N'-dicyclohexylcarbodiimide;FCCP;carbonylcyanide-p-trifluoromethoxyphenylhydrazon;→H+;quantity of acid equivalents translocated inwards;NEM;N-ethylmaleimide;ΔpH;pH difference across the membrane;PIPES;piperazine-N;N-bis-2-ethanesulphonic acid;Δψ;transmembrane potential