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 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.

Radiation inactivation analysis of H+‐pyrophosphatase from submitochondrial particles of etiolated mung bean seedlings

“…Proton translocation of submitochondrial particles was measured as fluorescence quenching of acridine orange (excitation 495 nm, emission 530 nm) by a Hitachi F‐4000 fluorescence spectrophotometer as previously described [4]. The reaction mixture (2 ml) contained 10 mM HEPES–Tris (pH 7.5), 50 mM KCl, 250 mM sucrose, 5 mM MgSO 4 , 1 mM EGTA, 5 μM acridine orange, and 50 μg submitochondrial particles.…”

Radiation inactivation analysis of thylakoid protein kinase systems in light and in darkness

“…Irradiation was performed with a 60 Co irradiator (approximately 1.11×10 6 GBq) and the dose rate was 7.00×10 -3 Mrad s -1 determined as previously described (Pan et al 1987, Wang et al 1988, Ma et al 1993, Tzeng et al 1996, Jiang et al 2000, Wu et al 2004. Internal standardization of radiation exposure was carried out with G 6 PDH.…”

Structural Studies of the Vacuolar H+-Pyrophosphatase: Sequence Analysis and Identification of the Residues Modified by Fluorescent Cyclohexylcarbodiimide and Maleimide

“…Highly purified H + -PPase reconstituted into proteoliposomes exhibits the H + -translocating activity (Sato et al 1994). The purified H + -PPase consists of a single polypeptide and forms a functionally active homodimer within the membrane (Maeshima 1990, Sato et al 1991, Tzeng et al 1996. Analysis of cDNAs encoding the H + -PPases from Arabidopsis thaliana and other plants has revealed that the H + -PPase consists of about 770 amino acid residues and has 12 to 15 putative membrane-spanning regions , Tanaka et al 1993, Zhen et al 1997.…”