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Preparation of sealed tonoplast and plasma-membrane vesicles from Catharanthus roseus (L.) G. Don. cells by free-flow electrophoresis

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Abstract

Highly purified tonoplast and plasmamembrane vesicles were isolated from microsomes of Catharanthus roseus (L.) G. Don. by preparative free-flow electrophoresis. The relative amounts of tonoplast and plasma-membrane vesicles in the total microsomes varied with the pH of the grinding medium. The most electronegative fractions were identified as tonoplast using nitrate-inhibited, azide-resistant Mg2+-ATPase and pyrophosphatase activities as enzyme markers. The least electronegative fractions were identified as plasma membrane using glucan-synthase-II and UDPG:sterolglucosyl-transferase activities as enzyme markers. Other membrane markers, latent inosine-5′-diphosphatase (Golgi), NADPH-cytochrome-c reductase (ER) and cytochrome-c oxidase (mitochondria) were recovered in the fractions intermediate between tonoplast and plasma membrane and did not contaminate either the tonoplast or the plasma-membrane fractions. In the course of searching for a reliable marker for tonoplast, the pyrophosphatase activity was found to be essentially associated with the tonoplast fractions purified by free-flow electrophoresis from C. roseus and other plant materials. The degree of sealing of the tonoplast and plasmamembrane vesicles was probed by their ability to pump protons (measurements of quinacrine quenching) and to generate a membrane potential (absorption spectroscopy of Oxonol VI). A critical evaluation of vesicles sidedness is presented.

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Abbreviations

CCCP:

carbonyl cyanide m-chlorophenylhydrazone

Con A:

concanavalin A

Cyt:

cytochrome

LysoPC:

lysophosphatidylcholine

Pi:

orthophoshate

PPiase:

pyrophosphatase

IDPase:

inosine-5′-diphosphatase

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Authors and Affiliations

  1. Centre de Physiologie Végétale de l'Université Paul Sabatier, URA CNRS n∘ 241, 118 Route de Narbonne, F-31062, Toulouse Cedex, France

    Hervé Canut, Sylvie Baudracco, Mireille Cabané, Alain-Michel Boudet & Gérard Marigo

Authors
  1. Hervé Canut
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  2. Sylvie Baudracco
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  3. Mireille Cabané
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  4. Alain-Michel Boudet
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  5. Gérard Marigo
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Additional information

We thank Pr. Robert Dargent and André Moisan (Laboratoire de Cryptogamie, Toulouse, France) for use of their electron-microscope facilities. This work was supported by the Centre National de la Recherche Scientifique and by a grant “Dynamique du fonctionnement de la vacuole” from the Ministère de la Recherche et de la Technologie.

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Canut, H., Baudracco, S., Cabané, M. et al. Preparation of sealed tonoplast and plasma-membrane vesicles from Catharanthus roseus (L.) G. Don. cells by free-flow electrophoresis. Planta 184, 448–456 (1991). https://doi.org/10.1007/BF00197892

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  • DOI: https://doi.org/10.1007/BF00197892

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