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Immunoaffinity purification and biochemical characterization of plasma membrane arabino-galactan-rich glycoproteins of Nicotiana glutinosa

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Abstract

Monoclonal antibody PN 16.4B4 reacts with an epitope present on the external face of the plasma membrane as shown by immunofluorescent staining of Nicotiana glutinosa L. protoplasts (Norman et al. 1986, Planta 167, 452–459). We show here that this epitope is present in a glycan moiety and defines a family of surface glycoproteins with molecular masses in the range 135–180 kilodalton (kDa). These glycoproteins are exclusively associated with the plasma membrane as demonstrated by immunostaining of highly purified plasma membrane vesicles obtained by aqueous two-phase partitioning of microsomal fractions. The bulk of these glycoproteins were not released by high-salt washing, sonication or hypotonie shock treatment of plasma membrane vesicles, demonstrating a tight association with the membrane. Triton X-114 partitioning of plasma membrane vesicles indicates that these antigens are hydrophilic, peripheral membrane glycoproteins. The glycoproteins were purified by immunoaffinity chromatography following solubilization in sodium dodecyl sulfate and shown to contain glycan moieties abundant in arabinose and galactose linked to a 50-kDa polypeptide rich in alanine, glycine, serine and threonine.

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Abbreviations

CHAPS:

3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate

ELISA:

enzyme-linked immunoabsorbent assay

kDa:

kilodalton

Mr :

relative molecular mass

NP-40:

nonidet P-40

SDS-PAGE:

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

TFMS:

trifluoromethane sulfonic acid

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Author notes

  1. Per Kjellbom

    Present address: Department of Plant Biochem, University of Lund, P.O.B. 124, S-221 00, Lund, Sweden

  2. Michael G. Hahn

    Present address: Complex Carbohydrate Research Center, University of Georgia, P.O.B. 5677, 30613, Athens, GA, USA

Authors and Affiliations

  1. Plant Biology Laboratory, Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, 92037, La Jolla, CA, USA

    Paul M. Norman, Per Kjellbom, Desmond J. Bradley, Michael G. Hahn & Christopher J. Lamb

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  1. Paul M. Norman
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  2. Per Kjellbom
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  3. Desmond J. Bradley
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  4. Michael G. Hahn
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  5. Christopher J. Lamb
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Additional information

We thank the University of Georgia-Department of Energy Complex Carbohydrate Research Center, Athens, USA for carbohydrate analysis, Mindy S. Fitter and David R. Lerner for helpful discussions, and Valerie Zatorski and Cynthia Doane for preparation of the manuscript. This research was supported by grants to C.J.L. from the McKnight Foundation, the Rockefeller Foundation, the Seaver Institute and U.S. Department of Agriculture, Competitive Grants Program (83-CRCR-1-1251). P.K. was supported by the Swedish Council for Forestry and Agricultural Research, D.B. by a NATO-Science and Engineering Research Council (UK) Fellowship.

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Norman, P.M., Kjellbom, P., Bradley, D.J. et al. Immunoaffinity purification and biochemical characterization of plasma membrane arabino-galactan-rich glycoproteins of Nicotiana glutinosa . Planta 181, 365–373 (1990). https://doi.org/10.1007/BF00195889

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  • DOI: https://doi.org/10.1007/BF00195889

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