Subunit interaction of vacuolar H+-pyrophosphatase as determined by high hydrostatic pressure.

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.

High-pressure effects on vacuolar H+-ATPase from etiolated mung bean seedlings.

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.

Isolation and partial purification of cytochrome oxidases from Bacillus thuringiensis subsp. israelensis HD-567.

Analyses of the cell membrane fractions by spectral absorbance revealed the presence of cytochrome c and three cytochrome oxidases in Bacillus thuringiensis subsp. israelensis HD-567-cytochromes a+a3, d, and o. A modified procedure was used to purify the cytochrome c:o complex from this organism. The oxidase complex was first solubilized from a sonic-disrupted cell membrane fraction (R3 fraction) using deoxycholate and KCl. The resulting soluble fraction was further purified by Sephadex G-50 gel filtration and DEAE ionexchange chromatography. TMPD oxidase specific activity and cytochrome concentration were assayed to monitor the purification procedures. The F7 fraction (obtained after G-50 chromatography) contained cytochrome c (0.44 nmole/mg protein), cytochrome a+a3 (0.26 nmole/mg protein), and cytochrome o (0.38 nmole/mg protein), which had 6-fold, 3.4-fold and 18.9-fold increase, respectively, by comparison with the original R3 fraction. The TMPD oxidase specific activity of the F7 fraction also increased 4.8 fold. The F8 fraction (obtained after the final DEAE chromatography) contained a cytochrome c:o complex only (cytochrome c, 0.46 nmole/mg protein; cytochrome o, 0.16 nmole/mg protein), but no cytochrome a+a3 was found. Both the TMPD oxidase specific activity and the cytochrome o were attenuated greatly in comparison with the F7 fraction. SDS-PAGE analysis revealed that the F7 fraction contained numerous protein components, while the F8 fraction contained only a prominent major protein of 113.5 kD thought to be the cytochrome c:o complex, and a minor polypeptide (MW = 65.8 kD). Although the final DEAE procedure removed many undesired polypeptides, TMPD oxidase activity and cytochrome o component were also lost greatly. Kinetic studies of this cytochrome c:o complex is in progress in our laboratory.