Brij 58, a polyoxyethylene acyl ether, creates membrane vesicles of uniform sidedness. A new tool to obtain inside‐out (cytoplasmic side‐out) plasma membrane vesicles

• Fredrik Johansson

• Malin Olbe

• Marianne Sommarin

• Christer Larsson

抄録

Most of the plasma membrane vesicles formed upon homogenization of plant tissue have a right‐side‐out (cytoplasmic side‐in) orientation. Subsequent purification of plasma membrane vesicles using aqueous two‐phase partitioning leads to a further enrichment in right‐side‐out vesicles resulting in preparations with 80–90% of the vesicles in this orientation. Thus, to be able to assay, e.g. the ion‐pumping activities of the H + ‐ATPase and the Ca 2+ ‐ATPase, which expose their active sites towards the cytoplasm, the vesicles have to be inverted. This is very efficiently achieved by including 0.05% of the detergent Brij 58 (C 16 E 20 ) in the assay medium, which produces 100% sealed, inside‐out (cytoplasmic side‐out) vesicles from preparations of 80–90% right‐side‐out vesicles. This was shown by assaying ATP‐dependent H + pumping using the ΔpH probe acridine orange and dissipating the H + gradient with nigericin, and by assaying ATP‐dependent Ca 2+ transport using 45 CA 2+ and dissipating the Ca 2+ gradient with the ionophore A23187. The presence of intact vesicles was confirmed by electronmicroscopy. The detergent Brij 58 is a polyoxyethylene acyl ether and a survey among some other members of this series revealed that those with a head group of relatively large size (E 20–23 ) showed this ‘non‐detergent behavior’, whereas those with smaller head groups (E 8–10 ) behaved as normal detergents and permeabilized the membranes. Thus, a very convenient system for studies on ion‐pumping activities and other vectorial properties of the plasma membrane is obtained by simply including the detergent Brij 58 in the assay medium.

収録刊行物

• The Plant Journal

  The Plant Journal 7 (1), 165-173, 1995-01

  Wiley

キーワード

• Cell Biology
• Plant Science
• Genetics

詳細情報

• CRID: 1361981470846132992
• NII論文ID: 30014607702
• DOI: 10.1046/j.1365-313x.1995.07010165.x
• ISSN: 1365313X
• ISSN: 09607412
• Web Site: https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1046%2Fj.1365-313X.1995.07010165.x
• Web Site: https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-313X.1995.07010165.x
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