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. 1976 Nov;73(11):3891–3895. doi: 10.1073/pnas.73.11.3891

Reconstitution of intramembrane particles in recombinants of erythrocyte protein band 3 and lipid: effects of spectrin-actin association.

J Yu, D Branton
PMCID: PMC431254  PMID: 1069273

Abstract

The integral membrane protein Band 3 of the human erythrocyte, either purified or in a crude Triton X-100 extract of ghosts, was combined with egg lecithin in a cholate solution. During dialysis to remove cholate, lipid bilayer vesicles formed in which Band 3 existed as a dimer and in which intramembrane particles indistinguishable from those in the native membrane were exposed by freeze-fracturing. The recombinant vesicles were stable in both high and low salt concentrations, sedimented at a density that increased in prportion to their protein content, and bound spectrin-actin extracted from erythrocyte ghosts. When spectrin-actin was associated with the vesicles, the behavior of the recombinant intramembrane particles simulated that of the erythrocyte ghost intramembrane particles: they were dispersed at pH 7.6 and aggregrated at pH 5-5.5. Thus, some of the characteristics of the native membrane have been reconstituted in the recombinant.

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Selected References

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

  1. Bretscher M. S. Membrane structure: some general principles. Science. 1973 Aug 17;181(4100):622–629. doi: 10.1126/science.181.4100.622. [DOI] [PubMed] [Google Scholar]
  2. Cabantchik Z. I., Rothstein A. Membrane proteins related to anion permeability of human red blood cells. I. Localization of disulfonic stilbene binding sites in proteins involved in permeation. J Membr Biol. 1974;15(3):207–226. doi: 10.1007/BF01870088. [DOI] [PubMed] [Google Scholar]
  3. Clarke S. The size and detergent binding of membrane proteins. J Biol Chem. 1975 Jul 25;250(14):5459–5469. [PubMed] [Google Scholar]
  4. DODGE J. T., MITCHELL C., HANAHAN D. J. The preparation and chemical characteristics of hemoglobin-free ghosts of human erythrocytes. Arch Biochem Biophys. 1963 Jan;100:119–130. doi: 10.1016/0003-9861(63)90042-0. [DOI] [PubMed] [Google Scholar]
  5. Elgsaeter A., Branton D. Intramembrane particle aggregation in erythrocyte ghosts. I. The effects of protein removal. J Cell Biol. 1974 Dec;63(3):1018–1036. doi: 10.1083/jcb.63.3.1018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Elgsaeter A., Shotton D. M., Branton D. Intramembrane particle aggregation in erythrocyte ghosts. II. The influence of spectrin aggregation. Biochim Biophys Acta. 1976 Feb 19;426(1):101–122. doi: 10.1016/0005-2736(76)90433-8. [DOI] [PubMed] [Google Scholar]
  7. FOLCH J., LEES M., SLOANE STANLEY G. H. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957 May;226(1):497–509. [PubMed] [Google Scholar]
  8. Fairbanks G., Steck T. L., Wallach D. F. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry. 1971 Jun 22;10(13):2606–2617. doi: 10.1021/bi00789a030. [DOI] [PubMed] [Google Scholar]
  9. Findlay J. B. The receptor proteins for concanavalin A and Lens culinaris phytohemagglutinin in the membrane of the human erythrocyte. J Biol Chem. 1974 Jul 25;249(14):4398–4403. [PubMed] [Google Scholar]
  10. Grant C. W., McConnell H. M. Glycophorin in lipid bilayers. Proc Natl Acad Sci U S A. 1974 Dec;71(12):4653–4657. doi: 10.1073/pnas.71.12.4653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ho M. K., Guidotti G. A membrane protein from human erythrocytes involved in anion exchange. J Biol Chem. 1975 Jan 25;250(2):675–683. [PubMed] [Google Scholar]
  12. Ji T. H., Nicolson G. L. Lectin binding and perturbation of the outer surface of the cell membrane induces a transmembrane organizational alteration at the inner surface. Proc Natl Acad Sci U S A. 1974 Jun;71(6):2212–2216. doi: 10.1073/pnas.71.6.2212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kahlenberg A. Partial puridication of a membrane protein from human erythrocytes involved in glucose transport. J Biol Chem. 1976 Mar 25;251(6):1582–1590. [PubMed] [Google Scholar]
  14. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  15. Lin S., Spudich J. A. Binding of cytochalasin B to a red cell membrane protein. Biochem Biophys Res Commun. 1974 Dec 23;61(4):1471–1476. doi: 10.1016/s0006-291x(74)80449-3. [DOI] [PubMed] [Google Scholar]
  16. Moor H. Recent progress in the freeze-etching technique. Philos Trans R Soc Lond B Biol Sci. 1971 May 27;261(837):121–131. doi: 10.1098/rstb.1971.0042. [DOI] [PubMed] [Google Scholar]
  17. Nicolson G. L., Painter R. G. Anionic sites of human erythrocyte membranes. II. Antispectrin-induced transmembrane aggregation of the binding sites for positively charged colloidal particles. J Cell Biol. 1973 Nov;59(2 Pt 1):395–406. doi: 10.1083/jcb.59.2.395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Pinto da Silva P., Douglas S. D., Branton D. Localization of A antigen sites on human erythrocyte ghosts. Nature. 1971 Jul 16;232(5307):194–196. doi: 10.1038/232194a0. [DOI] [PubMed] [Google Scholar]
  19. Reiss-Husson F. Structure des phases liquide-cristallines de différents phospholipides, monoglycérides, sphingolipides, anhydres ou en présence d'eau. J Mol Biol. 1967 May 14;25(3):363–382. doi: 10.1016/0022-2836(67)90192-1. [DOI] [PubMed] [Google Scholar]
  20. Segrest J. P., Gulik-Krzywicki T., Sardet C. Association of the membrane-penetrating polypeptide segment of the human erythrocyte MN-glycoprotein with phospholipid bilayers. I. Formation of freeze-etch intramembranous particles. Proc Natl Acad Sci U S A. 1974 Aug;71(8):3294–3298. doi: 10.1073/pnas.71.8.3294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Silva P. P., Nicolson G. L. Freeze-etch localization of concanavalin A receptors to the membrane intercalated particles of human erythrocyte ghost membranes. Biochim Biophys Acta. 1974 Sep 23;363(3):311–319. doi: 10.1016/0005-2736(74)90071-6. [DOI] [PubMed] [Google Scholar]
  22. Steck T. L. Cross-linking the major proteins of the isolated erythrocyte membrane. J Mol Biol. 1972 May 14;66(2):295–305. doi: 10.1016/0022-2836(72)90481-0. [DOI] [PubMed] [Google Scholar]
  23. Steck T. L., Dawson G. Topographical distribution of complex carbohydrates in the erythrocyte membrane. J Biol Chem. 1974 Apr 10;249(7):2135–2142. [PubMed] [Google Scholar]
  24. Steck T. L. The organization of proteins in the human red blood cell membrane. A review. J Cell Biol. 1974 Jul;62(1):1–19. doi: 10.1083/jcb.62.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Steck T. L., Yu J. Selective solubilization of proteins from red blood cell membranes by protein perturbants. J Supramol Struct. 1973;1(3):220–232. doi: 10.1002/jss.400010307. [DOI] [PubMed] [Google Scholar]
  26. Strapazon E., Steck T. L. Binding of rabbit muscle aldolase to band 3, the predominant polypeptide of the human erythrocyte membrane. Biochemistry. 1976 Apr 6;15(7):1421–1424. doi: 10.1021/bi00652a011. [DOI] [PubMed] [Google Scholar]
  27. Taverna R. D., Langdon R. G. D-glucosyl isothiocyanate, an affinity label for the glucose transport proteins of the human erythrocyte membrane. Biochem Biophys Res Commun. 1973 Sep 18;54(2):593–599. doi: 10.1016/0006-291x(73)91464-2. [DOI] [PubMed] [Google Scholar]
  28. Tillack T. W., Scott R. E., Marchesi V. T. The structure of erythrocyte membranes studied by freeze-etching. II. Localization of receptors for phytohemagglutinin and influenza virus to the intramembranous particles. J Exp Med. 1972 Jun 1;135(6):1209–1227. doi: 10.1084/jem.135.6.1209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wang K., Richards F. M. An approach to nearest neighbor analysis of membrane proteins. Application to the human erythrocyte membrane of a method employing cleavable cross-linkages. J Biol Chem. 1974 Dec 25;249(24):8005–8018. [PubMed] [Google Scholar]
  30. Wang K., Richards F. M. Reaction of dimethyl-3,3'-dithiobispropionimidate with intact human erythrocytes. Cross-linking of membrane proteins and hemoglobin. J Biol Chem. 1975 Aug 25;250(16):6622–6626. [PubMed] [Google Scholar]
  31. Yu J., Fischman D. A., Steck T. L. Selective solubilization of proteins and phospholipids from red blood cell membranes by nonionic detergents. J Supramol Struct. 1973;1(3):233–248. doi: 10.1002/jss.400010308. [DOI] [PubMed] [Google Scholar]
  32. Yu J., Steck T. L. Isolation and characterization of band 3, the predominant polypeptide of the human erythrocyte membrane. J Biol Chem. 1975 Dec 10;250(23):9170–9175. [PubMed] [Google Scholar]

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