Abstract
With the eventual aim of purifying a membrane transport system by using reconstitution of transport activity as an assay, I showed that if, after the erythrocyte membrane is solubilized in deoxycholate, the detergent is removed, membrane vesicles re-form which retain glucose-transport activity. They take up and release D-glucose in preference to L-glucose and the uptake and release are sensitive to Hg2+ and phloretin. Release of tracer D-glucose is competitively inhibited by transported sugars inside the vesicles and increased by unlabelling D-glucose in the outside medium. Uptake of tracer is increased so much by preloading vesicles with unlabelled transported sugars that the tracer is probably concentrated against a gradient. When the membrane is solubilized, two proteins that span the membrane can be separated, suggesting that it will be possible to fractionate the membrane before reconstitution.
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- BARTLETT G. R. Phosphorus assay in column chromatography. J Biol Chem. 1959 Mar;234(3):466–468. [PubMed] [Google Scholar]
- BLIGH E. G., DYER W. J. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911–917. doi: 10.1139/o59-099. [DOI] [PubMed] [Google Scholar]
- Bodemann H., Passow H. Factors controlling the resealing of the membrane of human erythrocyte ghosts after hypotonic hemolysis. J Membr Biol. 1972;8(1):1–26. doi: 10.1007/BF01868092. [DOI] [PubMed] [Google Scholar]
- 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]
- Johnson S. M. The effect of charge and cholesterol on the size and thickness of sonicated phospholipid vesicles. Biochim Biophys Acta. 1973 Apr 25;307(1):27–41. doi: 10.1016/0005-2736(73)90022-9. [DOI] [PubMed] [Google Scholar]
- Karlish S. J., Lieb W. R., Ram D., Stein W. D. Kinetic parameters of glucose efflux from human red blood cells under zero-trans conditions. Biochim Biophys Acta. 1972 Jan 17;255(1):126–132. doi: 10.1016/0005-2736(72)90014-4. [DOI] [PubMed] [Google Scholar]
- Kirkpatrick F. H., Gordesky S. E., Marinetti G. V. Differential solubilization of proteins, phospholipids, and cholesterol of erythrocyte membranes by detergents. Biochim Biophys Acta. 1974 Apr 29;345(2):154–161. doi: 10.1016/0005-2736(74)90254-5. [DOI] [PubMed] [Google Scholar]
- Meissner G., Fleischer S. Reconstitution of functional sarcoplasmic reticulum membrane vesicles. Methods Enzymol. 1974;32:475–481. doi: 10.1016/0076-6879(74)32047-2. [DOI] [PubMed] [Google Scholar]
- Miller D. M. The kinetics of selective biological transport. V. Further data on the erythrocyte-monosaccharide transport system. Biophys J. 1971 Nov;11(11):915–923. doi: 10.1016/S0006-3495(71)86263-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mueller T. J., Morrison M. The transmembrane proteins in the plasma membrane of normal human erythrocytes. J Biol Chem. 1974 Dec 10;249(23):7568–7573. [PubMed] [Google Scholar]
- Shapiro A. L., Viñuela E., Maizel J. V., Jr Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem Biophys Res Commun. 1967 Sep 7;28(5):815–820. doi: 10.1016/0006-291x(67)90391-9. [DOI] [PubMed] [Google Scholar]
- 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]