KAHLENBEWC, A., and DQLANSKY, D. Structural requirements of glucose for its binding to isolated human erythrocyte membranes. Can. J. Bischem.The inhibition by various derivatives of D-glucose of the stereospecific uptake of this sugar by isolated human erythrocyte membranes was determined. Single changes at each of the five hydroxyi groups of D-glucose, except for the 2-deoxy and 6-deoxy derivatives, resulted in a variable decrease in the binding afinity of the sugar which was dependent upon the nature and locus of the alteration. In addition, the presence of the pyranose ring oxygen is required for optimal binding. The largest decrease in afinity effected by a single change of any s f the three hydroxyl groups of D-glucose which participate in the binding reaction (i.e. those at carbons 1,3. and 4) was obtained by the removal or derivatization of the anomeric (C-l) hydroxyl. A similar decrease in binding afinity was obtained upon sulfur substitution of the ring oxygen. The results are interpreted as indicating that the binding of D-glucose to its erythrocyte membrane acceptor site is probably mediated by hydrogen bonds between this membrane component and the hydroxyl groups at carbons 1, 3, and 4 of D-gliucose and the pyranose ring oxygen.
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 changes in phospholipid composition sf the inner (cytoplasmic) surface of the human erythrocyte membrane resulting from the digestion of sealed inside-out vesicles with phospholipases A, and C were determined. Virtually all of the phosphatidylethanolamine and phosphatidylserine and 30-48%; of the phosphatidylcholine and sphingomyelin sf insideout vesicles were found to be accessible to enzyme hydrolysis. In contrast, all of the above phospholipids of unsealed ghosts were susceptible to phospholipolytic digestion. These results are a direct demoi~stration of an asymmetric distribution of phospholipids in the human erythrocyte membrane. KahHenberg, A., Walker, C. & Rohrlick, W. (1974) Evidence for an Asymmetric Distribution of PhosphoIipids in the Human Erythrocyte Membrane. Can. J. Biochem. 52, 803-886 Wous avons dCterminC les changements apportks dans la composition ghospholipidique B la surface intkrieure (cytoplasmiqile) de la membrane des Crytkrscytes humains suite k H a digestion par les phsspholipases As et C de vksicules morp-pkslogiquemerat inverskes et scellbes, Preque toutes les phssphatidylCthano1amines et les phosphatidylsCrines et 30-40CjI des phosphatidylcholiraes et des sphingomy6Enes des vCsisules inverskes sont touchCes par H'hydrolyse enzymatique. Au contraire, tous ces phospholigides sont susceptibles de digestion ghssgholipolytique dam les membranes strsmatiques non scelldes. Ces rCsuHtats dbmontrent direetement la distribution asymdtrique des phospholipides dans la membrane Crythrocytaire hurnaine. [Traduit par le jounral]lntrgeductim ghosts have provided evidence for an asym-~h~ action of group-specific labels and metrical distribution of membrane phospholipids phospholipases (4, 5 ) towards erythrocytes and (see Ref. 6). In contrast to cell ghosts9 the -
A sensitive method for the measurement of the stereospecific uptake of D-glUCOSe by isolated human erythrocyte membranes has been developed. The method is based on the difference in uptake of L-[' C]glucose and D-[ *H]glucose
The effects of insulin on glucose transport and dissimilation have been studied in the rat fat pad and in human omentum. Intracellular glucose could not be demonstrated in the non-diabetic rat fat pad, with or without insulin. Slices of omentum, obtained during surgery from diabetic and non-diabetic patients, were incubated in a bicarbonate medium with a glucose concentration of 5.6 mM. Omentum from non-diabetic patients had intracellular glucose while diabetic tissue had none. Diabetic tissue had a significantly lower glucose uptake. In both, insulin stimulated glucose uptake in inverse relation to the basal uptake; but for comparable basal uptakes, the response to insulin was decreased in diabetic tissue. Glucose phosphorylation in omental tissue from non-diabetic humans had an apparent Km of 1.10 m M and a Vmax of 1.44 mg/g hour.Insulin (0.1 units/ml), bovine or human serum albumin, or a combination of insulin and bovine albumin, each increased glucose utilization by slices of non-diabetic omentum without affecting the intracellular glucose content or the free fatty acid content. In omental homogenates (cell-free), insulin and bovine serum albumin each increased the glucose utilization. In the presence of albumin, insulin increased the utilization only after the addition of glucose-6-phosphate dehydrogenase and phosphohexose isomerase in amounts sufficient to establish that hexokinase was rate-limiting for glucose utilization.It is concluded that (A) in diabetic human omentum and in normal rat fat pad, transport is rate-limiting in glucose utilization; (B) insulin and albumin each stimulate both transport and phosphorylation in human omentum; (C) in human diabetes, the glucose transport process of omentum has a decreased basal rate and a decreased responsiveness to insulin.
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