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The isolation of coated vesicles from protoplasts of soybean

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Abstract

Fractions enriched in coated vesicles were obtained from protoplasts derived from suspension cultured Glycine max (L.) Merr. cells. Initial enrichment was achieved by isopycnic centrifugation of a protoplast homogenate through a linear sucrose gradient in a vertical rotor. The coated-vesicle fractions from this gradient were pooled and centrifuged through a second linear sucrose gradient in a rate zonal fashion to remove the larger contaminating membrane vesicles. The most prominent polypeptide in the coated-vesicle fractions, plant “clathrin”, had a relative molecular mass of approx. 190 kdalton as determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Other enriched polypeptides included bands at 105, 100, 96, 64, 50, 38 and 32 kdalton. This method was compared with a procedure utilizing sucrose step gradients for preparing coated vesicles from soybean protoplasts. The effectiveness of the isopycnic-rate zonal centrifugation procedure was also tested for the preparation of bovine-brain coated vesicles.

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Authors and Affiliations

  1. Plant Biotechnology Institute, National Research Council of Canada, S7N 0W9, Saskatoon, Sask., Canada

    Brent G. Mersey

  2. Department of Biology, University of Saskatchewan, S7N 0W0, Saskatoon, Sask., Canada

    Lawrence R. Griffing, Patricia J. Rennie & Larry C. Fowke

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  1. Brent G. Mersey
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  2. Lawrence R. Griffing
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  3. Patricia J. Rennie
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  4. Larry C. Fowke
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NRCC No. 23142

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Mersey, B.G., Griffing, L.R., Rennie, P.J. et al. The isolation of coated vesicles from protoplasts of soybean. Planta 163, 317–327 (1985). https://doi.org/10.1007/BF00395141

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  • DOI: https://doi.org/10.1007/BF00395141

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