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.
Glucose deprivation of chick embryo fibroblasts results in a cycloheximide-sensitive stimulation of hexose transport and an increase in the levels of glucose-regulated polypeptides of molecular weights 75 000 and 95 000. The relationship between these two phenomena is evaluated in this study. The glucose deprivation-induced stimulation of hexose transport was observed to occur in two phases: a rapid (complete by 15 min) cycloheximide-insensitive increase of 50-100% and a slower (observable by 6 h) cycloheximide-sensitive increase in transport to about five times the basal level. The time course of the latter increase preceded that of the appearance of the 75 000 and 95 000 dalton polypeptides; by the time that increases in the levels of these polypeptides were observed, the hexose uptake rates had almost reached their maximum value. Upon cellular fractionation, the greatest enrichment of the 75 000 and 95 000 dalton polypeptides was observed in the endoplasmic reticulum fraction, which was devoid of vesicular stereospecific D-glucose uptake activity. The plasma membrane fraction was enriched in stereospecific D-glucose uptake activity. The plasma membrane fraction was enriched in stereospecific D-glucose uptake activity but not in the 75 000 and 95 000 dalton polypeptides. The glucose deprivation-induced increase in hexose uptake was not prevented by tunicamycin, although this inhibitor of protein glycosylation decreased the hexose uptake of glucose-fed cells by 80% after 24 h. However, under these latter conditions an increase in the levels of the 75 000 and 95 000 dalton polypeptides was observed. On the basis of this data, we conclude that the polypeptides of molecular weights 75 000 and 95 000 are not involved in glucose transport.
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