N‐Ethylmaleimide Modifies the Conformation of the Plasma Membrane H+‐ATPase, Strengthening the Inhibitory Action of the C‐terminal Domain1

Abstract

Abstract: The effect of cysteine modification with N‐ethylmalei‐mide (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 (0.1–1mM) 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 W‐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.