Reconstitution of D-glucose transport in vesicles composed of lipids and intrinsic protein (Zone 4.5) of the human erythrocyte membrane
✍ Scribed by Kahlenberg, Arthur ;Zala, Cedric A.
- Publisher
- Wiley (John Wiley & Sons)
- Year
- 1977
- Tongue
- English
- Weight
- 870 KB
- Volume
- 7
- Category
- Article
- ISSN
- 0091-7419
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✦ Synopsis
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.