Abstract
Sucrose uptake into tonoplast vesicles, which were prepared from red beet (Beta vulgaris L.) vacuoles isolated by two different methods, was stimulated by MgATP. Using the same medium as for osmotic disruption of vacuoles, membrane vesicles were prepared from tissue homogenates of dormant red beet roots and separated by high-speed centrifugation through a discontinuous dextran gradient. A low-density microsomal fraction highly enriched in tonoplast vesicles could be further purified from contaminating ER vesicles by inclusion of 5 mM MgCl2 in the homogenization medium. These vesicles were able to transport sucrose in an ATP-dependent manner against a concentration gradient, whereas vesicles from regions of other densities lacked this feature, indicating that ATP stimulation of sucrose uptake took place only at the tonoplast membrane. Sucrose uptake was optimal at pH 7 in the presence of MgATP and could be stimulated by superimposed pH gradients (vesicle interior acidic) in the absence of MgATP, which is consistent with the operation of a sucrose/H+-antiporter at the tonoplast. Tonoplast vesicles, obtained in high yield from tissue homogenates of red beet roots, exhibited sugar-uptake characteristics comparable to those of intact vacuoles; these characteristics included similarities in K m (1.7 mM), sensitivity to inhibitors and specificity for sucrose.
Many experiments were carried out at the Experiment Station of the HSPA, Aiea, Hawaii and financed by an NSF grant to Dr. Maretzki and Mrs. M. Thom.
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Abbreviations
- CCCP:
-
carbonylcyanide m-chlorophenylhydrazone
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Part of this work was done in: Laboratoire de Physiologie Végétale, Prof Jean Guern, C.N.R.S., 15 Av. de la Terrasse, F-91190 Gif-sur-Yvette, France, and HSPA, Experiment Station, 99-193 Aiea Heights Drive, Aiea, Hawaii 96701, USA
I thank Dr. Andrew Maretzki (Aiea, Hawaii, USA) for critically reading and correcting the manuscript. Part of this work has been done in Professor Jean Guern's laboratory at the C.N.R.S. in Gif-sur-Yvette, France, and was made possible by a grant to the author by the Deutsche Forschungsgemeinschaft.
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Getz, H.P. Sucrose transport in tonoplast vesicles of red beet roots is linked to ATP hydrolysis. Planta 185, 261–268 (1991). https://doi.org/10.1007/BF00194069
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DOI: https://doi.org/10.1007/BF00194069