The influence of extracellular-side Ca2+ on the activity of the plasma membrane H+-ATPase from wheat roots

Abstract

Plasma membrane vesicles were purified from wheat roots by discontinuous sucrose gradient centrifugation and two-phase partitioning methods. The influence of extracellular-side Ca²⁺ on the activity of the plasma membrane H⁺ -ATPase from wheat roots was studied. The results showed that the ATP hydrolytic activities of the plasma membrane H⁺ -ATPase were inhibited by the cytoplasmic-side Ca²⁺. Within 0~200 µmol/L the ATPase activity decreased gradually with the increase in Ca²⁺ concentration; the ATPase activity was inhibited by 40% when Ca²⁺ concentration was 1000 µmol/L. However, the ATP hydrolytic activities were recovered by the presence of extracellular-side Ca²⁺. Results showed that the ATPase activities were increased with the increase in extracellular-side Ca²⁺; when the extracellular-side Ca²⁺ was 1000 µmol/L, the ATPase activity was recovered by 87.5%. Further studies found that the extracellular-side Ca²⁺ increased the DPH polarisation and decreased the MC540 fluorescence intensity, showing that membrane fluidity was decreased and membrane stacking was increased by the external Ca²⁺. The above results suggested that the plasma membrane H⁺ -ATPase could be regulated by the extracellular side Ca²⁺ through affecting the plasma membrane physical states.