PROPERTIES OF THE PROTON-PUMPING PYROPHOSPHATASE IN TONOPLAST VESICLES OF ACER-PSEUDOPLATANUS - FUNCTIONAL MOLECULAR-MASS AND POLYPEPTIDE COMPOSITION

The properties of a proton pumping pyrophosphatase were studied in pur e tonoplast preparations obtained from isolated vacuoles of Acer pseud oplatanus cells. A complex of Mg2+ and PPi was the specific substrate with an apparent Km for PPi of 33 muM. Inorganic pyrophosphate hydroly sis was coupled with proton transport. The H+-pyrophosphatase activity was stimulated by K+ and inhibited by Na+, F-, imidodiphosphate, meth ylenediphosphonate, diethylstilbestrol, thiol reagents and carbanilate derivatives. Permeant anions activated the PPi-dependent H+-pumping b y dissipating DELTAphi. The enzyme was insensitive to oligomycin, moly bdate and vanadate. The functional molecular mass analysed by radiatio n inactivation was found to be 88 +/- 8 kDa for substrate hydrolysis. The H+-pyrophosphatase was purified by gel filtration and subsequent i on exchange chromatography. Analysis by SDS-PAGE revealed one major po lypeptide of about 66 kDa which was an underestimated value of molecul ar mass because of its hydrophobicity as reported for other tonoplast PPases. This polypeptide cross-reacted with the polyclonal antibodies raised against the inorganic pyrophosphatase of mung bean vacuoles. Th ese results suggest that the tonoplast pyrophosphatase of A. pseudopla tanus is probably equivalent to tonoplast pyrophosphatases described i n other materials and particularly to the tonoplast PPase of Arabidops is thaliana which cDNA has been cloned and sequenced.