Reconstitution of D-glucose transport in vesicles composed of lipids and intrinsic protein (zone 4.5) of the human erythrocyte membrane.
Elucidation of the mechanism of facilitated D-glucose transport in human erythrocytes is dependent on the identification and isolation of the membrane protein(s) mediating this process. Based on the fact that stereospecific D-glucose transport is reconstituted in liposomes prepared by sonication of a lipid suspension with ghosts or fractions derived from ghosts, a quantitative assay for the stereospecific D-glucose transport activity of these fractions was developed (Zala CA, Kahlenberg A: Biochem Biophys Res Commun 72:866, 1976). This assay was used to monitor the purification of ghosts. The solubilized membrane protein fraction was chromatographed on a column of diethylaminoethyl cellulose which was eluted stepwise with NaCl-phosphate buffers of increasing ionic strength. A fraction, eluted at an ionic strength of 0.1, displayed a 13- and 27-fold increase in reconstituted transport activity relative to ghosts and to the unfractionated Triton X-100 extract, respectively. This fraction, when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, consisted predominantly of the ghost proteins with an apparent molecular weight of 55,000, commonly designated as zone 4.5; periodic acid-Schiff-sensitive membrane glycoproteins 1-4 were absent. Transport reconstituted by this preparation of zone 4.5 membrane proteins was almost completely abolished by 1-fluoro-2,4-dinitrobenzene, mercuric chloride, and p-chloromercuribenzene sulfonate, but was unaffected by sodium iodoacetate. Extra- and intraliposomal phloretin and cytochalasin B, respectively, exhibited partial inhibition. The stereospecificity and inhibition characteristics of the reconstituted transport imply that all the components of the erythrocyte D-glucose transport system are contained in the zone 4.5 membrane protein preparation.
D001693:Biological Transport, Active / Q000187:drug effects; D004910:Erythrocyte Membrane / Q000032:analysis / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008081:Liposomes / Q000378:metabolism; D008563:Membrane Lipids / Q000378:metabolism*; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity
D008081:Liposomes; D008563:Membrane Lipids; D008565:Membrane Proteins; D005947:Glucose