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 chromatogr...

Full description

Saved in:
Bibliographic Details
Published in:Biochemical journal 1996-05, Vol.316 ( Pt 1) (1), p.143-147
Main Authors: Tzeng, C M, Yang, C Y, Yang, S J, Jiang, S S, Kuo, S Y, Hung, S H, Ma, J T, Pan, R L
Format: Article
Language:English
Subjects:
Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone - pharmacology
Chromatography, Gel
Cobalt Radioisotopes
Dose-Response Relationship, Radiation
Fabaceae - enzymology
Gramicidin - pharmacology
Inorganic Pyrophosphatase
Kinetics
Macromolecular Substances
Models, Structural
Plants, Medicinal
Pyrophosphatases - antagonists & inhibitors
Pyrophosphatases - chemistry
Pyrophosphatases - radiation effects
Vacuoles - enzymology
Valinomycin - pharmacology
Citations: Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:0264-6021
1470-8728
DOI:10.1042/bj3160143