Detergent sensitivity of the tonoplast H(+)-ATPase and its purification from Beta vulgaris

Tonoplast membrane fractions were isolated from beetroot (Beta vulgaris) using a refined method of preparation which significantly improved the yield of active tonoplast H(+)-ATPase, and electron microscopy showed these fractions to be a preparation of small vesicles, of diameter 500 nm to 50 nm and a minor fraction consisting of mainly tubular membrane structures of diameter 5 nm and length up to 1 micron. The stability of the tonoplast H(+)-ATPase was assessed in the presence of many biological detergents, using a linked assay. The addition of detergent to tonoplast membranes generally led to an increase in ATPase activity, and activity was maintained in a wide range of both non-ionic and zwitterionic detergents. Using the non-ionic detergent dodecyl maltoside, the tonoplast H(+)-ATPase was partially purified using ion-exchange chromatography on an HPLC system. Very high rates of ATP hydrolysis were recorded in these fractions. The purified membranes behaved as expected in the presence of known activators and inhibitors. An unexpected observation, however, was that low concentrations of vanadate could significantly increase the rate of H(+)-ATPase activity.