title: High-pressure effects on vacuolar H+-ATPase from etiolated mung bean seedlings.
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 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.
authors.affiliation: Kao-Hsiung Medical College, Taiwan, Republic of China.
authors.forename: Y R
authors.initials: YR
authors.lastname: Tsai
authors.forename: S J
authors.initials: SJ
authors.lastname: Yang
authors.forename: S S
authors.initials: SS
authors.lastname: Jiang
authors.forename: S J
authors.initials: SJ
authors.lastname: Ko
authors.forename: S H
authors.initials: SH
authors.lastname: Hung
authors.forename: S Y
authors.initials: SY
authors.lastname: Kuo
authors.forename: R L
authors.initials: RL
authors.lastname: Pan
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D007887:Fabaceae / Q000201:enzymology*; D006868:Hydrolysis; D010946:Plants, Medicinal; D011312:Pressure; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014617:Vacuoles / Q000201:enzymology*
chemical_list: D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases