Vacuolar Chloride Transport in Mesembryanthemum crystallinum L. Measured Using the Fluorescent Dye Lucigenin

Vacuolar Chloride Transport in Mesembryanthemum crystallinum L. Measured Using the Fluorescent... To study vacuolar chloride (Cl−) transport in the halophilic plant Mesembryanthemum crystallinum L., Cl− uptake into isolated tonoplast vesicles was measured using the Cl−-sensitive fluorescent dye lucigenin (N,N′-dimethyl-9,9′-bisacridinium dinitrate). Lucigenin was used at excitation and emission wavelengths of 433 nm and 506 nm, respectively, and showed a high sensitivity towards Cl−, with a Stern-Volmer constant of 173 m −1 in standard assay buffer. While lucigenin fluorescence was strongly quenched by all halides, it was only weakly quenched, if at all, by other anions. However, the fluorescence intensity and Cl−-sensitivity of lucigenin was shown to be strongly affected by alkaline pH and was dependent on the conjugate base used as the buffering ion. Chloride transport into tonoplast vesicles of M. crystallinum loaded with 10 mm lucigenin showed saturation-type kinetics with an apparent K m of 17.2 mm and a V max of 4.8 mm min−1. Vacuolar Cl− transport was not affected by sulfate, malate, or nitrate. In the presence of 250 μm p-chloromercuribenzene sulfonate, a known anion-transport inhibitor, vacuolar Cl− transport was actually significantly increased by 24%. To determine absolute fluxes of Cl− using this method, the average surface to volume ratio of the tonoplast vesicles was measured by electron microscopy to be 1.13 × 107 m−1. After correcting for a 4.4-fold lower apparent Stern-Volmer constant for intravesicular lucigenin, a maximum rate of Cl− transport of 31 nmol m−2 sec−1 was calculated, in good agreement with values obtained for the plant vacuolar membrane using other techniques. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Membrane Biology Springer Journals

Vacuolar Chloride Transport in Mesembryanthemum crystallinum L. Measured Using the Fluorescent Dye Lucigenin

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Publisher
Springer Journals
Copyright
Copyright © Inc. by 2000 Springer-Verlag New York
Subject
Life Sciences; Biochemistry, general; Human Physiology
ISSN
0022-2631
eISSN
1432-1424
D.O.I.
10.1007/s002320010003
Publisher site
See Article on Publisher Site

Abstract

To study vacuolar chloride (Cl−) transport in the halophilic plant Mesembryanthemum crystallinum L., Cl− uptake into isolated tonoplast vesicles was measured using the Cl−-sensitive fluorescent dye lucigenin (N,N′-dimethyl-9,9′-bisacridinium dinitrate). Lucigenin was used at excitation and emission wavelengths of 433 nm and 506 nm, respectively, and showed a high sensitivity towards Cl−, with a Stern-Volmer constant of 173 m −1 in standard assay buffer. While lucigenin fluorescence was strongly quenched by all halides, it was only weakly quenched, if at all, by other anions. However, the fluorescence intensity and Cl−-sensitivity of lucigenin was shown to be strongly affected by alkaline pH and was dependent on the conjugate base used as the buffering ion. Chloride transport into tonoplast vesicles of M. crystallinum loaded with 10 mm lucigenin showed saturation-type kinetics with an apparent K m of 17.2 mm and a V max of 4.8 mm min−1. Vacuolar Cl− transport was not affected by sulfate, malate, or nitrate. In the presence of 250 μm p-chloromercuribenzene sulfonate, a known anion-transport inhibitor, vacuolar Cl− transport was actually significantly increased by 24%. To determine absolute fluxes of Cl− using this method, the average surface to volume ratio of the tonoplast vesicles was measured by electron microscopy to be 1.13 × 107 m−1. After correcting for a 4.4-fold lower apparent Stern-Volmer constant for intravesicular lucigenin, a maximum rate of Cl− transport of 31 nmol m−2 sec−1 was calculated, in good agreement with values obtained for the plant vacuolar membrane using other techniques.

Journal

The Journal of Membrane BiologySpringer Journals

Published: Oct 1, 2000

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