N-Ethylmaleimide Modifies the Conformation of the Plasma Membrane H⁺-ATPase, Strengthening the Inhibitory Action of the C-terminal Domain*

Abstract

The effect of cysteine modification with N-ethylmaleimide (NEM) on the activity of the plasma membrane (PM) H⁺-ATPase and on its activation state was investigated in PM isolated from aged red beet parenchyma slices. Treatment of PM with increasing concentrations of NEM (0.1 - 1 mM) drastically reduced H⁺-ATPase activity. The inhibiting effect of PM treatment with NEM was stronger when the H⁺-ATPase activity was assayed at pH values (7.1 - 7.2) higher than that optimal for enzyme activity (6.3). If the PM H⁺-ATPase was activated by proteolytic cleavage of the C-terminal domain or by its displacement by fusicoccin prior to NEM treatment, the inhibitory effect of NEM on the H⁺-ATPase activity became independent of the pH of the assay medium. Moreover, inhibition by NEM of H⁺-ATPase activity also became independent of the pH of the assay medium if the C-terminal was proteolytically cleaved or displaced by lysophosphatidylcholine after NEM treatment of the PM. Controlled trypsin treatment of NEM-treated PM produced, beside the 90 kDa truncated PM H⁺-ATPase, fragments of 60 to 30 kDa of the enzyme that were undetectable after trypsin treatment of control PM. These results indicate that PM treatment with NEM modifies the H⁺-ATPase conformation, exposing trypsin cleavage sites scarcely accessible in control PM and strengthening the autoinhibitory action of the C-terminal domain.

Key words

Plasma membrane H⁺-ATPase - N-ethylmaleimide - red beet - Beta vulgaris