SUBUNIT INTERACTION OF VACUOLAR H-PYROPHOSPHATASE AS DETERMINED BY HIGH HYDROSTATIC-PRESSURE()

Vacuolar H+-pyrophosphatase (H+-PPase) from etiolated hypocotyls of mu ng 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 prote in-concentration-dependent manner. The V-max value of vacuolar H+-PPas e was dramatically decreased by pressurization from 293.9 to 70.2 mu m ol of PP, (pyrophosphate) consumed/h per mg of protein, while the K-m, value decreased from 0.35 to 0.08 mM, implying that the pressure trea tment 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 revea ls high pressure treatment of H+-PPase provokes the subunit dissociati on from an active into inactive form. High hydrostatic pressure also i nduces the conformational change of vacuolar H+-PPase as determined by spectroscopic techniques. Our results indicate the importance of prot ein-protein interaction for this novel proton-translocating enzyme. Wo rking models are proposed to interpret the pressure inactivation of va cuolar H+-PPase. We also suggest that association of identical subunit s of vacuolar H+-PPase is not random but proceeds in a specific manner .