Molecular Markers for Ion Compartmentation in Cells of Higher Plants 1. Isolation of vacuoles of high-purity

Leach, R P, Rogers, J, Wheeler, K P, Flowers, T J and Yeo, A R (1990) Molecular Markers for Ion Compartmentation in Cells of Higher Plants 1. Isolation of vacuoles of high-purity. Journal of Experimental Botany, 41 (9). pp. 1079-1087. ISSN 0022-0957

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Abstract

The tonoplast plays a crucial role in ion compartmentation, which is a central feature of the salt tolerance of halophytes, but we do not know the properties of the membrane that confer this ability. A method was, therefore, developed for the isolation of vacuoles from Suaeda maritima (L.) Dum. of sufficiently high purity to enable biochemical characterization of their lipid and protein composition. Tonoplast fractions produced by density gradient centrifugation, as well as vacuoles isolated by a variety of methods (including DEAE dextran lysis, digitonin lysis, and mechanical shear forces) were unacceptably contaminated. A highly pure vacuole preparation was obtained when protoplasts were lysed by a mild hypotonic shock in alkaline buffer, in the presence of the compatible cytosolute glycine-betaine, followed by shear force during ultracentrifugation; cytoplasmic contamination was prevented by the addition of the zwitterionic detergent 3-([3-cholamidopropyl]dimethylammonio)-l-propanesulphonate (CHAPS). Light microscopy of this preparation revealed no intact protoplasts and no contamination by chlorophyll could be detected. Electron microscopy showed the vacuoles to be single-membrane-bound structures, and was the only criterion upon which vacuoles could be separated reliably from vacuoplasts, in which the plasmalemma is collapsed on to the tonoplast. Analysis by SDS-PAGE showed that a total of 15 polypeptides were enriched in the tonoplast and 27 in the soluble fraction from vacuole preparations, with a pattern similar to that reported for glycophytic species. The pure tonoplast exhibited both vanadate-insensitive ATPase and pyrophosphatase activities, but the properties of these enzymes were broadly similar to those of glycophytes. Analysis of membrane fatty acids showed that the degree of saturation of the putative tonoplast preparation increased as the assessment of the purity of the preparation (made by microscopy) increased. The ATPase could be substantially purified by ion-exchange FPLC. The results are discussed in relation to the degree of purity needed in membrane preparations in order to be suitable for biochemical analysis.

Item Type: Article
Additional Information: Times Cited: 13 Leach, rp rogers, wj wheeler, kp flowers, tj yeo, ar
Schools and Departments: School of Life Sciences > Evolution, Behaviour and Environment
Subjects: Q Science
Depositing User: Catrina Hey
Date Deposited: 22 May 2012 09:41
Last Modified: 30 Nov 2012 17:12
URI: http://sro.sussex.ac.uk/id/eprint/39054
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