Subunit structure of vacuolar proton-pyrophosphatase as determined by radiation inactivation.
Vacuolar proton-pyrophosphatase (H(+)-PPase) of mung bean seedlings contains a single kind of polypeptide with a molecular mass of approx. 73 kDa. However, in this study, a molecular mass of approx. 140 kDa was obtained for the purified vacuolar H(+)-PPase by size-exclusion gel-filtration chromatography, suggesting that the solubilized form of this enzyme is a dimer. Radiation inactivation analysis of tonoplast vesicles yielded functional masses of 141.5 +/- 10.8 and 158.4 +/- 19.5 kDa for PP1 hydrolysis activity and its supported proton translocation respectively. These results confirmed the in situ dimeric structure of the membrane-bound H(+)-PPase of plant vacuoles. Further target-size analysis showed that the functional unit of purified vacuolar H(+)-PPase was 71.1 +/- 6.7 kDa, indicating that only one subunit of the purified dimeric complex would sufficiently display its enzymic reaction. Moreover, in the presence of valinomycin and KCl, the functional size of membrane-bound H(+)-PPase was decreased to approx. 63.4 +/- 6.3 kDa. A working model was proposed to elucidate the structure of native H(+)-PPase on vacuolar membrane as a functional dimer. Factors that would disturb the membrane, e.g. membrane solubilization and the addition of valinomycin and KCl, may induce an alteration in its enzyme structure, subsequently resulting in a different functional size.
Tzeng
C M
CM
Institute of Radiation Biology, College of Nuclear Science, National Tsing Hua University, Hsin Chu, Taiwan, Republic of China.
Yang
C Y
CY
Yang
S J
SJ
Jiang
S S
SS
Kuo
S Y
SY
Hung
S H
SH
Ma
J T
JT
Pan
R L
RL
D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002850:Chromatography, Gel; D003037:Cobalt Radioisotopes; D004307:Dose-Response Relationship, Radiation; D007887:Fabaceae / Q000201:enzymology*; D006096:Gramicidin / Q000494:pharmacology; D043564:Inorganic Pyrophosphatase; D007700:Kinetics; D046911:Macromolecular Substances; D008961:Models, Structural; D010946:Plants, Medicinal; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000528:radiation effects; D014617:Vacuoles / Q000201:enzymology; D014634:Valinomycin / Q000494:pharmacology
D003037:Cobalt Radioisotopes; D046911:Macromolecular Substances; D006096:Gramicidin; D014634:Valinomycin; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase