Regulation ofVacuolar pHofPlant Cells 11.A 31PNMR StudyoftheModifications ofVacuolar pH inIsolated Vacuoles Induced byProtonPumpingandCation/H+ Exchanges

Thevacuolar pHandthetrans-tonoplast ApHmodifications induced bytheactivity ofthetwoproton pumpsH+-ATPase and H -PPase andbytheproton exchanges catalyzed bytheNa+/H+ andCa2+/H+ antiports atthetonoplast ofisolated intact vacuoles prepared fromCatharanthus roseuscells enriched ininorganic phosphate (YMathieu etal1988Plant Physiol [in press]) were measured using the31PNMRtechnique. TheH+-ATPase induced anintravacuolar acidification aslarge as0.8pHunit, building a trans-tonoplast ApHupto2.2pHunits. Thehydrolysis ofthe phosphorylated substrate andthevacuolar acidification were monitored simultaneously toestimate kinetically theapparent stoichiometry between thevectorial protonpumping andthe hydrolytic activity oftheH+-ATPase. A ratio ofH+translocated/ ATPhydrolyzed of1.97± 0.06(mean± standard error) was calculated. Pyrophosphate-treated vacuoles werealsoacidified