Vacuolar H+-pyrophosphatase (H+-PPase) from etiolated hypocotyls of mung bean (Vigna radiata L.) is a homodimer with a molecular mass of 145 kDa. The vacuolar H+-PPase was subjected to high hydrostatic pressure to investigate its structure and function. The inhibition of H+-PPase activity by high hydrostatic pressure has a pressure-, time- and protein-concentration-dependent manner. The Vmax value of vacuolar H+-PPase was dramatically decreased by pressurization from 293.9 to 70.2 micromol of PPi (pyrophosphate) consumed/h per mg of protein, while the Km value decreased from 0.35 to 0.08 mM, implying that the pressure treatment increased the affinity of PPi to vacuolar H+-PPase but decreased its hydrolysis. The physiological substrate and its analogues enhance high pressure inhibition of vacuolar H+-PPase. The HPLC profile reveals high pressure treatment of H+-PPase provokes the subunit dissociation from an active into inactive form. High hydrostatic pressure also induces the conformational change of vacuolar H+-PPase as determined by spectroscopic techniques. Our results indicate the importance of protein-protein interaction for this novel proton-translocating enzyme. Working models are proposed to interpret the pressure inactivation of vacuolar H+-PPase. We also suggest that association of identical subunits of vacuolar H+-PPase is not random but proceeds in a specific manner.
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 Vmax and Km of solubilized vacuolar H+-ATPase, implying an increase in ATP binding affinity, but a decrease in the ATP hydrolysis activity. Physiological substrate, Mg2+-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.
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