Publications

• Comparative studies on the electrical properties of the H+ translocating ATPase and pyrophosphatase of the vacuolar-lysosomal compartment.

  Hedrich R., Kurkdjian A., Guern J., Flugge U.I.

  The electrical properties of the vacuolar-lysosomal H+ pumps were studied by direct measurement of the pump currents using the whole-cell configuration of the patch-clamp technique. Both pumps, the proton-translocating ATPase and pyrophosphatase, when activated by MgATP or inorganic Mg pyrophospha ... >> More

  The electrical properties of the vacuolar-lysosomal H+ pumps were studied by direct measurement of the pump currents using the whole-cell configuration of the patch-clamp technique. Both pumps, the proton-translocating ATPase and pyrophosphatase, when activated by MgATP or inorganic Mg pyrophosphate (MgPP(i)), transport protons into the vacuole and polarize the membrane potential (positive inside the vacuole). Accumulation of protons in the lumen of vacuole vesicles was monitored by absorbance changes of the pH probe, acridine orange. The electrochemical gradient provided by both the ATPase and pyrophosphatase stimulates effectively the uptake of various metabolites such as malate, citrate and sucrose. The maximal current density produced by the ATPase was about 2.5 microA/cm2 and about 0.5 microA/cm2 for the pyrophosphatase. K(m)ATP was 0.6 mM; K(m)PPi was 15-20 microM with progressive inhibition above 150 microM. At a cytoplasmic pH of 7.5 both enzymes were capable of pumping protons against a 10,000-fold concentration gradient (pH 3.5 inside the vacuole). Proton current produced by the ATPase was blocked reversibly by extravacuolar NO(3)- only. << Less

  EMBO J 8:2835-2841(1989) [PubMed] [EuropePMC]

• Chromatographic resolution of h-translocating pyrophosphatase from h-translocating ATPase of higher plant tonoplast.

  Rea P.A., Poole R.J.

  Membrane vesicles derived from the tonoplast of Beta vulgaris L. possess two predominant phosphohydrolase activities: a Cl(-)-stimulated, NO(3) (-)-inhibited ATPase, and a K(+)-stimulated, Na(+)-inhibited inorganic pyrophosphatase (PPase). The solubilization of tonoplast vesicles with 2% (w/v) Tri ... >> More

  Membrane vesicles derived from the tonoplast of Beta vulgaris L. possess two predominant phosphohydrolase activities: a Cl(-)-stimulated, NO(3) (-)-inhibited ATPase, and a K(+)-stimulated, Na(+)-inhibited inorganic pyrophosphatase (PPase). The solubilization of tonoplast vesicles with 2% (w/v) Triton X-100 containing 4 millimolar MgCl(2) and 1 millimolar ethylenediamine tetracetic acid, as protectants, gives high yields of both the ATPase and PPase in soluble form. Chromatography of the solubilized membranes on Sephacryl-400 results in the separation of the two enzymes. The PPase and ATPase are purified 4- and 17-fold, respectively, with quantitative recovery. The separated enzymes show negligible activity towards the other's substrate and the separated PPase only hydrolyzes pyrophosphate. The separated enzymes show mineral ion requirements identical to those of the corresponding pump and hydrolytic activities in native tonoplast and both solubilized enzymes are subject to phospholipid activation. << Less

  Plant Physiol 81:126-129(1986) [PubMed] [EuropePMC]

• Vacuolar H⁺-Pyrophosphatase and Cytosolic Soluble Pyrophosphatases Cooperatively Regulate Pyrophosphate Levels in <i>Arabidopsis thaliana</i>.

  Segami S., Tomoyama T., Sakamoto S., Gunji S., Fukuda M., Kinoshita S., Mitsuda N., Ferjani A., Maeshima M.

  Inorganic pyrophosphate (PPi) is a phosphate donor and energy source. Many metabolic reactions that generate PPi are suppressed by high levels of PPi. Here, we investigated how proper levels of cytosolic PPi are maintained, focusing on soluble pyrophosphatases (AtPPa1 to AtPPa5; hereafter PPa1 to ... >> More

  Inorganic pyrophosphate (PPi) is a phosphate donor and energy source. Many metabolic reactions that generate PPi are suppressed by high levels of PPi. Here, we investigated how proper levels of cytosolic PPi are maintained, focusing on soluble pyrophosphatases (AtPPa1 to AtPPa5; hereafter PPa1 to PPa5) and vacuolar H⁺-pyrophosphatase (H⁺-PPase, AtVHP1/FUGU5) in <i>Arabidopsis thaliana</i> In planta, five PPa isozymes tagged with GFP were detected in the cytosol and nuclei. Immunochemical analyses revealed a high abundance of PPa1 and the absence of PPa3 in vegetative tissue. In addition, the heterologous expression of each PPa restored growth in a soluble PPase-defective yeast strain. Although the quadruple knockout mutant plant <i>ppa1 ppa2 ppa4 ppa5</i> showed no obvious phenotypes, H⁺-PPase and PPa1 double mutants (<i>fugu5 ppa1</i>) exhibited significant phenotypes, including dwarfism, high PPi concentrations, ectopic starch accumulation, decreased cellulose and callose levels, and structural cell wall defects. Altered cell arrangements and weakened cell walls in the root tip were particularly evident in <i>fugu5 ppa1</i> and were more severe than in <i>fugu5</i> Our results indicate that H⁺-PPase is essential for maintaining adequate PPi levels and that the cytosolic PPa isozymes, particularly PPa1, prevent increases in PPi concentrations to toxic levels. We discuss <i>fugu5 ppa1</i> phenotypes in relation to metabolic reactions and PPi homeostasis. << Less

  Plant Cell 30:1040-1061(2018) [PubMed] [EuropePMC]

• Purification of an h-translocating inorganic pyrophosphatase from vacuole membranes of red beet.

  Sarafian V., Poole R.J.

  An H(+)-translocating inorganic pyrophosphatase (PPase) was isolated and purified from red beet (Beta vulgaris L.) tonoplast. One major polypeptide of molecular weight 67 kilodalton copurified with fluoride-inhibitable PPase activity when subjected to one-dimensional polyacrylamide gel electrophor ... >> More

  An H(+)-translocating inorganic pyrophosphatase (PPase) was isolated and purified from red beet (Beta vulgaris L.) tonoplast. One major polypeptide of molecular weight 67 kilodalton copurified with fluoride-inhibitable PPase activity when subjected to one-dimensional polyacrylamide gel electrophoresis. Overall, a 150-fold purification of the PPase was obtained, from the tonoplast fraction, through anion exchange chromatography of the detergent-solubilized membranes followed by ammonium sulfate precipitation and gel filtration chromatography. The purified polypeptide showed no cross-reactivity with antibodies raised against the 67 kilodalton subunit of the tonoplast ATPase. << Less

  Plant Physiol 91:34-38(1989) [PubMed] [EuropePMC]