Properties of sugar and proton transport at the tomato fruit tonoplast
Properties of sugar and proton transport at the tomato fruit tonoplast
Tonoplast vesicles were prepared from tomato (Lycopersicon esculentum L.) pericarp and purified on a discontinuous Dextran gradient. ATPase hydrolytic activity was inhibited by nitrate and bafilomycin A1, but was insensitive to vanadate and azide. PPase hydrolytic activity was inhibited by NaF but was insensitive to nitrate, bafilomycin A1, vanadate and azide. H+-PPase H+-pumping and PPi-hydrolysis activities were optimal at pH 7.0. Kinetic studies of PPase activity revealed an apparent Km for PPi of 14 mmol m-1. Identical distributions of bafilomycin- and NO3--sensitive ATPase activities within continuous density gradients confirmed the potential of bafilomycin-sensitive ATPase activity as a marker assay for the tonoplast. By comparing the distribution of bafilomycin-sensitive ATPase activity with the distribution of PPase activity it was possible to locate the PPase enzyme exclusively at the tonoplast. The apparent density of the tonoplast did not change during fruit development. Measurements of tonoplast PPase and ATPase activities during tomato fruit development over a 35 day period revealed an 80% reduction in PPase specific activity together with a small decrease in ATPase specific activity. ATP- or PPi-dependent ΔpH generation was measured by monitoring the quenching of quinacrine fluorescence. No H^+ efflux could be measured by the addition of sucrose to energized vesicles. The data obtained suggested that a H^+/sucrose antiport may not be the mechanism of sucrose uptake at the tonoplast. Similar results were obtained using glucose, fructose and sorbitol.
The lack of ATP stimulation of [^14C]-sucrose uptake again suggested that uptake into the vacuole does not involve a H^+/sucrose antiport. Uptake rates of radiolabelled sucrose were very similar to those of glucose and fructose indicating a non-specific uptake mechanism. Although sucrose and hexose uptake was concentration dependent, no clear saturation kinetics were observed at sugar concentrations up to 100 mol m^-3. Inhibition of sucrose and hexose uptake by p-chloromercuribenzene sulphonate (PCMBS) did however implicate the involvement of a carrier.
University of Southampton
1993
Milner, Ian David
(1993)
Properties of sugar and proton transport at the tomato fruit tonoplast.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
Tonoplast vesicles were prepared from tomato (Lycopersicon esculentum L.) pericarp and purified on a discontinuous Dextran gradient. ATPase hydrolytic activity was inhibited by nitrate and bafilomycin A1, but was insensitive to vanadate and azide. PPase hydrolytic activity was inhibited by NaF but was insensitive to nitrate, bafilomycin A1, vanadate and azide. H+-PPase H+-pumping and PPi-hydrolysis activities were optimal at pH 7.0. Kinetic studies of PPase activity revealed an apparent Km for PPi of 14 mmol m-1. Identical distributions of bafilomycin- and NO3--sensitive ATPase activities within continuous density gradients confirmed the potential of bafilomycin-sensitive ATPase activity as a marker assay for the tonoplast. By comparing the distribution of bafilomycin-sensitive ATPase activity with the distribution of PPase activity it was possible to locate the PPase enzyme exclusively at the tonoplast. The apparent density of the tonoplast did not change during fruit development. Measurements of tonoplast PPase and ATPase activities during tomato fruit development over a 35 day period revealed an 80% reduction in PPase specific activity together with a small decrease in ATPase specific activity. ATP- or PPi-dependent ΔpH generation was measured by monitoring the quenching of quinacrine fluorescence. No H^+ efflux could be measured by the addition of sucrose to energized vesicles. The data obtained suggested that a H^+/sucrose antiport may not be the mechanism of sucrose uptake at the tonoplast. Similar results were obtained using glucose, fructose and sorbitol.
The lack of ATP stimulation of [^14C]-sucrose uptake again suggested that uptake into the vacuole does not involve a H^+/sucrose antiport. Uptake rates of radiolabelled sucrose were very similar to those of glucose and fructose indicating a non-specific uptake mechanism. Although sucrose and hexose uptake was concentration dependent, no clear saturation kinetics were observed at sugar concentrations up to 100 mol m^-3. Inhibition of sucrose and hexose uptake by p-chloromercuribenzene sulphonate (PCMBS) did however implicate the involvement of a carrier.
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Published date: 1993
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Local EPrints ID: 462252
URI: http://eprints.soton.ac.uk/id/eprint/462252
PURE UUID: 1417089c-3bd6-44f6-aaa3-732721a47539
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Date deposited: 04 Jul 2022 19:04
Last modified: 04 Jul 2022 19:04
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Author:
Ian David Milner
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