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Isolation and characterization of epinectin, a novel adhesion protein for epithelial cells.

Abstract
A 70,000-mol-wt protein was isolated from A431 carcinoma cell extracellular matrix that promotes cell substratum adhesion of these epidermoid tumor cells. Extracellular matrix was isolated by a modification of a procedure described by Hedman et al. (Hedman, K., M. Kurkinen, K. Alitalo, A. Vaheri, S. Johansson, and M. Höök, 1979 J. Cell Biol., 81:83-91) and Yamada and Weston (Yamada, K., and J. A. Weston, 1974, Proc. Natl. Acad. Sci. USA, 71:3492-3496). Cells were solubilized with 0.5% deoxycholate, 10 mM Tris, 0.9% NaCl, and 1 mM phenylmethylsulfonyl fluoride, pH 8.0. The residual matrix was then removed from the plates with 6 M urea and 1 mM phenylmethylsulfonyl fluoride and phosphate-buffered saline. SDS PAGE gels of the 6 M urea extract showed one major band at 70,000-mol-wt by Coomassie Blue staining. A 70,000-mol-wt isotopically-labeled band could also be extracted from the matrix of cells incubated with [35S]methionine. Because of the presence of this protein on squamous-derived epithelial cells we have called the 70,000-mol-wt molecule epinectin. Indirect immunofluorescence with polyclonal rabbit antibodies against epinectin stained A431 cells pericellularly in dense punctate accumulations and along the plasma membrane. Enzyme-linked immunoassays and gel-transfer immunolocalization studies showed that the extract did not cross-react with antibodies to fibronectin, laminin, serum-spreading factor, epibolin, or keratin. Additionally, antibodies to epinectin did not cross-react with these proteins. Further studies showed that epinectin does not bind to gelatin. Cell-adhesion assay, using radiolabeled A431 carcinoma cells on various adhesion-promoting substrates, showed that epinectin has similar adhesion-promoting capacity as serum-spreading factor, was somewhat less active than fibronectin, but more effective than laminin or epibolin. Epinectin appears to be a unique protein isolated from epidermoid tumor cells that is distinct from other known adhesion proteins.
AuthorsJ Enenstein, L T Furcht
JournalThe Journal of cell biology (J Cell Biol) Vol. 99 Issue 2 Pg. 464-70 (Aug 1984) ISSN: 0021-9525 [Print] UNITED STATES
PMID6378923 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)
Chemical References
  • Extracellular Matrix Proteins
  • Immune Sera
  • Neoplasm Proteins
  • epinectin
Topics
  • Carcinoma, Squamous Cell
  • Cell Adhesion
  • Cell Line
  • Enzyme-Linked Immunosorbent Assay
  • Epithelium (ultrastructure)
  • Extracellular Matrix Proteins
  • Female
  • Fluorescent Antibody Technique
  • Humans
  • Immune Sera
  • Molecular Weight
  • Neoplasm Proteins (isolation & purification)
  • Vulvar Neoplasms

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