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.
Yang
S J
SJ
Institute of Radiation Biology, College of Nuclear Science, National Tsing Hua University, Hsin Chu 30043, Taiwan, Republic of China.
Ko
S J
SJ
Tsai
Y R
YR
Jiang
S S
SS
Kuo
S Y
SY
Hung
S H
SH
Pan
R L
RL
D002462:Cell Membrane / Q000201:enzymology; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D007887:Fabaceae / Q000648:ultrastructure*; D005454:Fluorescence Polarization; D006868:Hydrolysis; D006874:Hydrostatic Pressure; D043564:Inorganic Pyrophosphatase; D007700:Kinetics; D008970:Molecular Weight; D010946:Plants, Medicinal; D011755:Pyrophosphatases / Q000737:chemistry* / Q000378:metabolism*; D013053:Spectrophotometry; D014617:Vacuoles / Q000201:enzymology*
D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase