Improved mathematical model for estimating H+ influx and H+ efflux in plant vacuolar vesicles acidified by ATPase or pyrophosphatase.
To adapt to environmental changes, plant cells very likely possess a biochemical system, using vacuoles, for maintaining cytoplasmic pH homeostasis. A simple approach is to estimate the active H(+) influx and H(+) efflux of isolated vacuolar vesicles, although there is no good mathematical model to describe H(+) flux. To establish a new quantitative model, vacuolar vesicles were isolated from hypocotyls of mung bean (Vigna radiata L.), and pyrophosphate (PPi)- or ATP-dependent acidification was monitored using acridine orange. The change of pH inside the vesicles (pH(in)) was calculated using a pH calibration curve relating fluorescence quenching with DeltapH. After formation of a steady state DeltapH, passive H(+) efflux was monitored after terminating pumping with ethylenediaminetetraacetate, and the relative H(+) permeability coefficient (p(H+)) was calculated. The H(+) efflux simulated using the p(H+) corresponded to the H(+) efflux determined experimentally. H(+) influx was then calculated by subtracting the predicted H(+) efflux from the experimental net H(+) influx. H(+) influx into vesicles driven by H(+)-PPase or H(+)-ATPase decreased exponentially as the intravesicular pH(in) decreased, suggesting modulation of pumping by DeltapH, pH(in), or both. Finally, the PPi- or ATP-dependent H(+) accumulation determined experimentally was closely simulated by the predicted H(+) influx and H(+) efflux. The ability to predict H(+) flux under different conditions provides a powerful tool for studying pH homeostasis.
Kawamura
Yukio
Y
Department of Cell Biology & Molecular Genetics, University of Maryland, HJ Patterson Hall, College Park, MD 20742-5815, USA. ykawa@iwate-u.ac.jp
D000165:Acridine Orange; D002463:Cell Membrane Permeability; D004492:Edetic Acid / Q000737:chemistry; D007887:Fabaceae / Q000201:enzymology*; D005456:Fluorescent Dyes / Q000737:chemistry; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D018546:Hypocotyl / Q000201:enzymology; D008962:Models, Theoretical; D011755:Pyrophosphatases / Q000032:analysis / Q000378:metabolism*; D013997:Time Factors; D025262:Vacuolar Proton-Translocating ATPases / Q000032:analysis / Q000378:metabolism*; D014617:Vacuoles / Q000201:enzymology*
D005456:Fluorescent Dyes; D006859:Hydrogen; D004492:Edetic Acid; D011755:Pyrophosphatases; D025262:Vacuolar Proton-Translocating ATPases; D000165:Acridine Orange