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 hy...

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Bibliographic Details
Published in:Biochemical journal 1998-04, Vol.331 (2), p.395-402
Main Authors: Yang, S.J, Ko, S.J, Tsai, Y.R, Jiang, S.S, Kuo, S.Y, Hung, S.H, Pan, R.L
Format: Article
Language:English
Subjects:
Cell Membrane - enzymology
Chromatography, Gel
Chromatography, High Pressure Liquid
Circular Dichroism
Dimerization
Electrophoresis, Polyacrylamide Gel
Fabaceae - ultrastructure
Fluorescence Polarization
Hydrolysis
Hydrostatic Pressure
Inorganic Pyrophosphatase
Kinetics
Molecular Weight
Plants, Medicinal
protein subunits
Pyrophosphatases - chemistry
Pyrophosphatases - metabolism
Spectrophotometry
Vacuoles - enzymology
Vigna radiata
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Summary: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.
ISSN:0264-6021
1470-8728
DOI:10.1042/bj3310395