Skip to main content
SpringerLink
Log in

Purification of GP57, and auxin-regulated extracellular glycoprotein of carrots, and its immunocytochemical localization in dermal tissues

  • Published:
Planta Aims and scope Submit manuscript

Abstract

A glycoprotein (GP57) was purified by ion-exchange and hydroxylapatite column chromatography from the 70%-ethanol precipitate of culture medium of non-embryogenic carrot cells (Daucus carota L.) grown with 2,4-dichlorophen-oxyacetic acid (2,4-D). Its apparent molecular mass (M r) was estimated to be 57000 by sodium dodecylsulfate-polyacrylamide gel electrophoresis and 50000 by gel filtration. GP57 contained 14% (w/w) carbohydrate; the M r of the peptide portion was estimated to be 55000 after deglycosylation by trifluoromethanesulfonic acid. GP57 is composed of two polypeptides with the same Mr and with very similar amino-acid composition but different pI values, 8.8 and 9.5. Both are rich in aspartic acid, serine and threonine, and may possess N-linked oligosaccharide chains, including fucose and xylose. A monoclonal antibody (MAb) against the purified GP57 reacted with both the pI 8.8 and the 9.5 components, as well as the deglycosylated GP57. Immunoblotting with the MAb indicated that GP57 is synthesized in and released from cultured cells which have been supplied with auxin. In immunocytochemical studies, GP57 was found in the space between the embryo and the endosperm of dry seeds, and its content decreased during germination. GP57 was also found in the endodermis and epidermis of young roots, the periderm of mature taproots, and the epidermis of petioles and young leaves.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

2,4-D:

2,4-dichlorophenoxyacetic acid

GP57:

M r-57000 glycoprotein

GP65:

M r-65000 glycoprotein

MAb:

monoclonal antibody

M r :

apparent molecular mass

SDS-PAGE:

sodium dodecylsulfate-polyacrylamide gel electrophoresis

TFMS:

trifluoromethanesulfonic acid

References

  • Albersheim, P., Nevins, D.J., English, P.D., Karr, A. (1967) A method for the analysis of sugars in plant cell-wall polysaccharides by gas-liquid chromatography. Carbohydr. Res. 5, 340–345

    Google Scholar 

  • Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254

    Google Scholar 

  • Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F. (1956) Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356

    Google Scholar 

  • Edge, A.S.B., Faltynek, C.R., Hof, L., Reichert, L.E., Weber, P. (1981) Deglycosylation of glycoproteins by trifluoromethanesulfonic acid. Anal. Biochem. 118, 131–137

    Google Scholar 

  • Esau, K. (1940) Developmental anatomy of the fleshy storage organ of Daucus carota. Hilgardia 13, 175–226

    Google Scholar 

  • Esau, K., (1977) Anatomy of seed plants. John Wiley & Sons, New York, USA

    Google Scholar 

  • Gefter, M.L., Margulies, D.H., Scharff, M.D. (1977) A simple method for polyethylene glycol-promoted hybridization of mouse myeloma cells. Somat. Cell Genet. 3, 231–236

    Google Scholar 

  • Gershoni, J.M., Palade, G.E. (1982) Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to a positively charged membrane filter. Anal. Biochem. 124, 396–405

    Google Scholar 

  • Goding, J.W. (1980) Antibody production by hybridomas. J. Immunol. Meth. 39, 285–308

    Google Scholar 

  • Ishihara, H., Takahashi, J., Oguri, S., Tejima, S. (1979) Complete structure of the carbohydrate moiety of Stem Bromelain. An application of the almond glycopeptidase for structural studies of glycopeptides. J. Biol. Chem. 254, 10715–10719

    Google Scholar 

  • Kijimoto-Ochiai, S., Katagiri, Y.U., Ochiai, H. (1985) Analysis of N-linked oligosaccharide chains of glycoproteins on nitrocellulose sheets using lectin-peroxidase reagents. Anal. Biochem. 147, 222–229

    Google Scholar 

  • Kolattukudy, P.E. (1984) Biochemistry and function of cutin and suberin. Can. J. Bot. 62, 2918–2933

    Google Scholar 

  • Konat, G., Offner, H., Mellah, J. (1984) Improved sensitivity for detection and quantitation of glycoproteins on polyacrylamide gels. Experientia 40, 303–304

    Google Scholar 

  • Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685

    Google Scholar 

  • Maruo, F., Okada, M. (1987) Monoclonal antibodies against Drosophila ovaries: their reaction with ovarian and embryonic antigens. Cell Different. 20, 45–54

    Google Scholar 

  • Matsubara, H., Sasaki, R.M. (1969) High recovery of tryptophan from acid hydrolysates of protein. Biochem. Biophys. Res. Commun. 35, 175–181

    Google Scholar 

  • McLean, I.W., Nakane, P.K. (1974) Periodate-lysine-paraformaldehyde fixative. A new fixative for immunoelectron microscopy. J. Histochem. Cytochem. 22, 1077–1083

    Google Scholar 

  • Murashige, T., Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473–497

    Google Scholar 

  • Ouchterlony, O., Nilsson, L.A. (1973) Iminunodiffusion and immunoelectrophoresis. In: Handbook of Experimental Immunology vol. 1, pp. 19.1–19.39, (Weir, D.M., ed J.B. Lippincott Co., Philadelphia, Penn., USA

    Google Scholar 

  • Piller, V., Piller, F., Cartron, J.-P. (1986) Isolation and characterization of an N-acetylgalactosamine specific lectin from Salvia sclarea seeds. J. Biol. Chem. 261, 14069–14075

    Google Scholar 

  • Prigent, M.-J. (1984) Preliminary investigation of the structure of the carbohydrate component of Vicia graminea lectin, a plant glycoprotein. Carbohydr. Res. 131, 83–92

    Google Scholar 

  • Satoh, S., Kamada, H., Harada, H., Fujii, T. (1986) Auxincontrolled glycoprotein release into the medium of embryogenic carrot cells. Plant Physiol. 81, 931–933

    Google Scholar 

  • Satoh, S., Satoh, E., Watanabe, T., Fujii, T. (1985) Isolation and characterization of a protease inhibitor from spinach leaves. Phytochemistry 24, 419–423

    Google Scholar 

  • Shibata, S., Goldstein, I.J., Baker, D.A. (1982) Isolation and characterization of a Lewis b-active lectin from Griffonia simplicifolia seeds. J. Biol. Chem. 257, 9324–9329

    Google Scholar 

  • Steward, F.C., Mapes, M.O., Smith, J. (1958) Growth and organized development of cultured cells. I. Growth and division of freely suspended cells. Am. J. Bot. 45, 693–703

    Google Scholar 

  • Streefkerk, J.G. (1972) Inhibition of erythrocyte pseudoperoxidase activity by treatment with hydrogen peroxide following methanol. J. Histochem. Cytochem. 20, 829–831

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Biological Sciences, University of Tsukuba, 305, Tsukuba, Ibarakl, Japan

    Shinobu Satoh & Tadashi Fujii

Authors
  1. Shinobu Satoh
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tadashi Fujii
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Satoh, S., Fujii, T. Purification of GP57, and auxin-regulated extracellular glycoprotein of carrots, and its immunocytochemical localization in dermal tissues. Planta 175, 364–373 (1988). https://doi.org/10.1007/BF00396342

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00396342

Key words

Search

Navigation