Reconstitution of D‐glucose transport in vesicles composed of lipids and intrinsic protein (Zone 4.5) of the human erythrocyte membrane

Abstract

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.

The influence of lipid composition on the barrier properties of band 3-containing lipid vesicles

“…These results are in agreement with previous studies, concerning the permeability characteristics of D-glucose carrier containing model systems [34,35]. Recently, it has been suggested that the 251 native D-glucose transporter of the human erythrocyte membrane is an accompanying component of band 3 and that band 4.5 represents a partially active fragment of the native transporter [36].…”

“…Chemicals and radiochemicals used were from the following sources: Na35SO4 (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40) (10.7 Ci/nmol) from New England Nuclear. Stractan (arabino galactan) from Sigma.…”

Developmental changes in glucose transport of guinea pig erythrocytes.

“…The first of these possibilities was eliminated by our finding that although liposomes prepared from Triton extracted newborn cells could preferentially take up D-glucose, no such binding was observed when Triton extracts from adult erythrocytes were used. The actual disappearance of the glucose carrier molecule was demonstrated by showing that the quantity of proteins in zone 4.5, the region of the electrophoretic pattern that manifests glucosebinding characteristics (1,2), diminishes markedly with maturation of guinea pigs. Lesser decreases were observed in bands seven and eight.…”

“…Recent studies ofthis system have suggested that subunits of the carrier protein are located in the area designated as zone 4.5 on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)1 (1)(2)(3). The erythrocytes ofmost newborn mammals also have the capacity to facilitate the transport of glucose, but in some species the permeability of erythrocytes to glucose is rapidly lost with maturation (4,5).…”

Identification of the stereospecific hexose transporter from starved and fed chicken embryo fibroblasts.

“…Photoaffinity labeling of the D-glucose transporter in the human erythrocyte by this method has established its validity (17). This D-glucose transporter has previously been identified and purified as a Mr 55,000 polypeptide (18)(19)(20) Cell Culture. Chicken embryo fibroblasts were prepared from 10-day-old embryos by modification of described methods (21).…”