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. 1982 Feb 1;201(2):287–295. doi: 10.1042/bj2010287

Isolation and characterization of plasma-membrane glycoproteins from pig epidermis

Ian A King 1,*, Anne Tabiowo 1,*
PMCID: PMC1163642  PMID: 7082290

Abstract

1. Non-desmosomal plasma membranes enriched in plasma-membrane marker enzymes and in metabolically labelled glycoproteins were isolated on a large scale from up to 500g of pig ear skin slices. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and periodic acid/Schiff staining revealed the presence of four major glycosylated components in the apparent molecular-weight range 150000–80000. 2. A large proportion of the marker enzymes, the d-[3H]glucosamine-labelled glycoproteins and the periodic acid/Schiff-stained glycoproteins were solubilized by 1% (w/v) sodium deoxycholate. However, several non-glycosylated proteins, in particular those with mol.wts. 81000, 41000 and 38000 (possibly cytoskeletal components), were relatively resistant to solubilization. 3. The deoxycholate-solubilized membranes were fractionated by lectin affinity chromatography using both concanavalin A–Sepharose 4B and lentil lectin–Sepharose 4B. From 75 to 85% of the applied glycoprotein was recovered from the columns. From 30 to 40% of the recovered glycoprotein was specifically bound by the lectins and was eluted with 2% (w/v) α-methyl d-mannoside. The enrichment of labelled glycoproteins in the material bound by the lectins (2.5-fold) was similar with both lectins, although the yield was somewhat greater when lentil lectin was used. The glycoprotein-enriched fraction was also enriched in all the plasma-membrane marker enzymes, indicating their probable glycoprotein nature. 4. The glycoprotein-enriched fraction contained the four major periodic acid/Schiff-stained bands that were detected in the original plasma membrane. They had apparent mol.wts. 147000, 130500, 108000 and 91400. The higher-molecular-weight components contained relatively more d-[3H]glucosamine, indicating differences in the sugar composition or in the metabolic turnover of the individual glycoproteins in culture. The material bound by the lectins also contained a number of lower-molecular-weight Coomassie Brilliant Blue-stained components. These were weakly stained by periodic acid/Schiff reagent and were lightly labelled with d-[3H]glucosamine, indicating that they contained less carbohydrate than the four major glycoprotein bands. 5. Chloroform/methanol-extracted plasma membranes and isolated glycoproteins had a similar carbohydrate composition, containing sialic acid, hexosamine, fucose, xylose, mannose, galactose and glucose. Glucose was not enriched in the isolated glycoproteins, suggesting that it may be a contaminant. Xylose, however, was enriched in the isolated glycoproteins. It remains to be established whether this sugar, which is not usually found in plasma-membrane glycoproteins, is a genuine constituent of plasma-membrane glycoproteins in the epidermis.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. ADACHI K. Metabolism of glycogen in the skin and the effect of x-rays. J Invest Dermatol. 1961 Nov;37:381–395. [PubMed] [Google Scholar]
  2. Allan D., Auger J., Crumpton M. J. Glycoprotein receptors for concanavalin A isolated from pig lymphocyte plasma membrane by affinity chromatography in sodium deoxycholate. Nat New Biol. 1972 Mar 8;236(62):23–25. doi: 10.1038/newbio236023a0. [DOI] [PubMed] [Google Scholar]
  3. Brown W. R., Barclay A. N., Sunderland C. A., Williams A. F. Identification of a glycophorin-like molecule at the cell surface of rat thymocytes. Nature. 1981 Feb 5;289(5797):456–460. doi: 10.1038/289456a0. [DOI] [PubMed] [Google Scholar]
  4. Clamp J. R. Analysis of glycoproteins. Biochem Soc Symp. 1974;(40):3–16. [PubMed] [Google Scholar]
  5. Drochmans P., Freudenstein C., Wanson J. C., Laurent L., Keenan T. W., Stadler J., Leloup R., Franke W. W. Structure and biochemical composition of desmosomes and tonofilaments isolated from calf muzzle epidermis. J Cell Biol. 1978 Nov;79(2 Pt 1):427–443. doi: 10.1083/jcb.79.2.427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gray G. M., King I. A., Yardley H. J. The plasma membrane of Malpighian cells from pig epidermis: isolation and lipid and protein composition. Br J Dermatol. 1980 Nov;103(5):505–515. doi: 10.1111/j.1365-2133.1980.tb01665.x. [DOI] [PubMed] [Google Scholar]
  7. Hartree E. F. Determination of protein: a modification of the Lowry method that gives a linear photometric response. Anal Biochem. 1972 Aug;48(2):422–427. doi: 10.1016/0003-2697(72)90094-2. [DOI] [PubMed] [Google Scholar]
  8. Hayman M. J., Crumpton M. J. Isolation of glycoproteins from pig lymphocyte plasma membrane using Lens culinaris phytohemagglutinin. Biochem Biophys Res Commun. 1972 May 26;47(4):923–930. doi: 10.1016/0006-291x(72)90581-5. [DOI] [PubMed] [Google Scholar]
  9. Hudson J. E., Johnson T. C. The degradation and turnover of fucosylated glycoproteins in the plasma membrane of a neuroblastoma-cell line. Biochem J. 1977 Aug 15;166(2):217–223. doi: 10.1042/bj1660217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hunt R. C., Ellar D. J. Isolation of the plasma membrane of a trypanosomatid flagellate: general characterisation and lipid composition. Biochim Biophys Acta. 1974 Mar 15;339(2):173–189. doi: 10.1016/0005-2736(74)90316-2. [DOI] [PubMed] [Google Scholar]
  11. King I. A., Tabiowo A. Effect of tunicamycin on epidermal glycoprotein and glycosaminoglycan synthesis in vitro. Biochem J. 1981 Aug 15;198(2):331–338. doi: 10.1042/bj1980331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. King I. A., Tabiowo A., Williams R. H. Incorporation of l-[3H]fucose and d-[3H]glucosamine into cell-surface-associated glycoconjugates in epidermis of cultured pig skin slices. Biochem J. 1980 Jul 15;190(1):65–77. doi: 10.1042/bj1900065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kornfeld R., Kornfeld S. Comparative aspects of glycoprotein structure. Annu Rev Biochem. 1976;45:217–237. doi: 10.1146/annurev.bi.45.070176.001245. [DOI] [PubMed] [Google Scholar]
  14. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. Long V. J., Yardley H. J. Phospholipids in cultured guinea pig skin. J Invest Dermatol. 1970 Feb;54(2):174–177. doi: 10.1111/1523-1747.ep12258437. [DOI] [PubMed] [Google Scholar]
  16. Lotan R., Nicolson G. L. Purification of cell membrane glycoproteins by lectin affinity chromatography. Biochim Biophys Acta. 1979 Dec 20;559(4):329–376. doi: 10.1016/0304-4157(79)90010-8. [DOI] [PubMed] [Google Scholar]
  17. Mescher M. F., Jose M. J., Balk S. P. Actin-containing matrix associated with the plasma membrane of murine tumour and lymphoid cells. Nature. 1981 Jan 15;289(5794):139–144. doi: 10.1038/289139a0. [DOI] [PubMed] [Google Scholar]
  18. Nozaki Y., Tanford C. The solubility of amino acids and two glycine peptides in aqueous ethanol and dioxane solutions. Establishment of a hydrophobicity scale. J Biol Chem. 1971 Apr 10;246(7):2211–2217. [PubMed] [Google Scholar]
  19. Olson M. O., Liener I. E. Some physical and chemical properties of concanavalin A, the phytohemagglutinin of the jack bean. Biochemistry. 1967 Jan;6(1):105–111. doi: 10.1021/bi00853a018. [DOI] [PubMed] [Google Scholar]
  20. Skerrow C. J. Intercellular adhesion and its role in epidermal differentiation. Invest Cell Pathol. 1978 Jan-Mar;1(1):23–37. [PubMed] [Google Scholar]
  21. Skerrow C. J., Matoltsy A. G. Chemical characterization of isolated epidermal desmosomes. J Cell Biol. 1974 Nov;63(2 Pt 1):524–530. doi: 10.1083/jcb.63.2.524. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Skerrow C. J., Matoltsy A. G. Isolation of epidermal desmosomes. J Cell Biol. 1974 Nov;63(2 Pt 1):515–523. doi: 10.1083/jcb.63.2.515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ugel A. R., Chrambach A., Rodbard D. Fractionation and characterization of an oligomeric series of bovine keratohyalin by polyacrylamide gel electrophoresis. Anal Biochem. 1971 Oct;43(2):410–426. doi: 10.1016/0003-2697(71)90271-5. [DOI] [PubMed] [Google Scholar]
  24. WARREN L. The thiobarbituric acid assay of sialic acids. J Biol Chem. 1959 Aug;234(8):1971–1975. [PubMed] [Google Scholar]
  25. Yasuda Y., Takahashi N., Murachi T. The composition and structure of carbohydrate moiety of stem bromelain. Biochemistry. 1970 Jan 6;9(1):25–32. doi: 10.1021/bi00803a004. [DOI] [PubMed] [Google Scholar]

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