Subunit structure of vacuolar proton-pyrophosphatase as determined by radiation inactivation

Abstract

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 PPi 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.