Abstract
A high-hydrostatic-pressure technique was employed to study the structure-function relationship of plant vacuolar H + -ATPase from etiolated mung bean seedlings (Vigna radiata L.). When isolated vacuolar H + -ATPase was subjected to hydrostatic pressure, the activity of ATP hydrolysis was markedly inhibited in a time-, protein concentration- and pressure-dependent manner. The pressure treatment decreased both V max and K m of solubilized vacuolar H + -ATPase, implying an increase in ATP binding affinity, but a decrease in the ATP hydrolysis activity. Physiological substrate, Mg 2+ -ATP, augmented the loss of enzymatic activity upon pressure treatment. However, ADP, AMP, and Pi exerted substantial protective effects against pressurization. Steady-state ATP hydrolysis was more sensitive to pressurization than single-site ATPase activity. The inactivation of solubilized vacuolar H + -ATPase by pressure may result from changes in protein-protein interaction. The conformational change of solubilized vacuolar H + -ATPase induced by hydrostatic pressure was further determined by spectroscopic techniques. The inhibition of vacuolar H + -ATPase under pressurization involved at least two steps. Taken together, our work indicates that subunit-subunit interaction is crucial for the integrity and the function of plant vacuolar H + -ATPase. It is also suggested that the assembly of the vacuolar H + -ATPase complex is probably not random, but follows a sequestered pathway. © 1998 Plenum Publishing Corporation.