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. 1990 Jun;93(2):412–417. doi: 10.1104/pp.93.2.412

Proton Gradient Across the Tonoplast of Riccia fluitans as a Result of the Joint Action of Two Electroenzymes 1

Eva Johannes 1,2, Hubert Felle 1
PMCID: PMC1062527  PMID: 16667481

Abstract

Using pH-sensitive microelectrodes (in vitro) and acridine orange photometry (in vivo), the actions of the two tonoplast phosphatases, the tp-ATPase and the tp-PPase, were investigated with respect to how effectively they could generate a transtonoplast pH-gradient. Under standard conditions the vacuoles of the aquatic liverwort Riccia fluitans have an in vivo pH of 4.7 to 5.0. In isolated vacuoles a maximal vacuolar pH (pHv) of 4.74 ± 0.1 is generated in the presence of 0.1 millimolar PPi, but only 4.93 ± 0.13 in the presence of 2.5 millimolar ATP. Both substrates added together approximate the value for PPi. Cl−-stimulates the H+-transport driven by the tp-ATPase, but has no effect on the tp-PPase. The transport activity of the tp-ATPase approximates saturation kinetics (K½ ≈ 0.5 millimolar), whereas transport by the tp-PPase yields an optimum around 0.1 millimolar PPi. The transtonoplast pH-gradient is dissipated slowly by weak bases, from which a vacuolar buffer capacity of roughly 300 to 400 millimolar/pHv unit has been estimated. From the free energy (−11.42 kilojoules per mole) for the hydrolysis of PPi under the given experimental conditions, we conclude that the PPase-stoichiometry (transported H+ per hydrolyzed substrate molecule) must be 1, and that in vivo this enzyme works as a H+-pump rather than as a pyrophosphate synthetase.

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Selected References

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