Plant Physiol. Drug Metab Dispos
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Plant Physiology 94:1149-1156 (1990)
© 1990 American Society of Plant Biologists

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Membranes and Bioenergetics

Tonoplast Vesicles of Opposite Sidedness from Soybean Hypocotyls by Preparative Free-Flow Electrophoresis 1

Hervé Canut, Andrew Brightman, Alain M. Boudet and D. James Morré

Department of Medicinal Chemistry and Pharmacognosy, Purdue University, West Lafayette, Indiana 47907, Centre de Physiologie Végétale de l'Université Paul Sabatier, U.A. CNRS No. 241, 118, Route de Narbonne, F-31062 Toulouse Cedex, France

Tonoplast vesicles were purified from a microsomal fraction isolated from etiolated soybean hypocotyls (Glycine max L.) by preparative free-flow electrophoresis. Marker enzyme determinations and immunoblot analysis against the vacuolar-ATPase confirmed the nature and the purity of the isolated membranes. A purified tonoplast fraction also was obtained by consecutive sucrose and glycerol centrifugation which was further resolved into two different populations of vesicles (TA and TB) by free-flow electrophoresis. The determination of the sidedness of these different vesicles included concanavalin A binding as an imposed label, NADH-ferricyanide oxidoreductase cytochemistry, and ATPase latency. The tonoplast fractions, obtained by consecutive sucrose and glycerol gradient centrifugations, were found to consist of a mixture of two populations of vesicles of opposite sidedness. The least electronegative fraction obtained by free-flow electrophoresis (TB) consisted predominantly of cytoplasmic side out tonoplast vesicles while a fraction of greater electronegativity (TA) contained the cytoplasmic side in tonoplast vesicles. The relative amounts of each type of vesicle varied with the method of homogenization. Razor blade chopping, Polytron, and Waring Blendor homogenization gave predominantly cytoplasmic side out vesicles, whereas mashing with a mortar and pestle gave nearly equal amounts of the two populations of membrane vesicles of different orientation.

1 Work supported in part by a grant from the National Institutes of Health CA 36761 and by a fellowship to H. C. from the National Science Foundation/Centre National de la Recherche Scientifique Exchange Program.






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