Reconstitution of D-glucose transport in vesicles composed of lipids and intrinsic protein (zone 4.5) of the human erythrocyte membrane (original) (raw)
Related papers
Biochemical and Biophysical Research Communications, 1976
The stereospecific efflux of D-glucose from liposomes formed upon sonication of erythrocyte lipid with various membrane fractions was measured in order to assess their D-glucose transport activity. Extrinsic and intrinsic membrane proteins were separated into soluble and membrane-bound fractions, respectively by extraction of ghosts with 2,3-dimethylmaleic anhydride. Reconstitution of D-glucose transport was catalyzed only by the intrinsic membrane proteins. Upon subsequent Triton X-lO0 extraction of these proteins, reconstitution of D-glucose transport was associated with both the extract and membrane residue separated by high-speed centrifugation. The membrane residue did not contain any periodic acid-Schiff-sensitive glycoprotein and consisted chiefly of a protein component of 95,000 molecular weight (Band 3), a portion of which was present in the Triton X-lO0 extract. These results support the proposal that Band 3 proteins are directly involved in D-glucose transport.
The stereospecific d-glucose transport activity of cholate extracts from human erythrocyte membranes
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1981
The glucose transport protein of human erythrocyte membranes was solubilized with cholate to facilitate rapid reconstitution and direct glucose transport measurements. This may simplify the isolation of the native glucose transporter. In most experiments the membranes were prepared from fresh blood within 8 h, frozen in liquid nitrogen and stored at -70°C to minimize proteolytie degradation. Solubilization with 25 mM cholate in the presence of 200 mM NaCI at pH 8.4 for 12 min at room temperature gave a high D-glucose transport activity. The solubilized mixture contained 20% of the total membrane protein, only 6% of the polypeptides of molecular weight around 90 000, 23% of the polypeptides of molecular weight around 55 000, 30% of the phospholipids and at least 6% of the stereospecific D-glucose transport activity. At cholate concentrations up to 22 mM the ratio of solubilized phospholipids to cholate increased steeply, concomitant with an increase in solubilized activity. Above 30 mM cholate the activity diminished. At 4°C the activity of the extract decreased rapidly within the first day and slowly during the next few days. The initial changes seem to have produced a fairly stable, but not native form or fragment of the transporter. When 20 mM EDTA and 5 mM dithioerythritol were included in the solubilization mixture a high activity was preserved for about one day.
Protides of the biological fluids, 1982
The glucose transport protein of human erythrocyte membranes was solubilized with cholate to facilitate rapid reconstitution and direct glucose transport measurements. This may simplify the isolation of the native glucose transporter. In most experiments the membranes were prepared from fresh blood within 8 h, frozen in liquid nitrogen and stored at-70°C to minimize proteolytie degradation. Solubilization with 25 mM cholate in the presence of 200 mM NaCI at pH 8.4 for 12 min at room temperature gave a high D-glucose transport activity. The solubilized mixture contained 20% of the total membrane protein, only 6% of the polypeptides of molecular weight around 90 000, 23% of the polypeptides of molecular weight around 55 000, 30% of the phospholipids and at least 6% of the stereospecific D-glucose transport activity. At cholate concentrations up to 22 mM the ratio of solubilized phospholipids to cholate increased steeply, concomitant with an increase in solubilized activity. Above 30 mM cholate the activity diminished. At 4°C the activity of the extract decreased rapidly within the first day and slowly during the next few days. The initial changes seem to have produced a fairly stable, but not native form or fragment of the transporter. When 20 mM EDTA and 5 mM dithioerythritol were included in the solubilization mixture a high activity was preserved for about one day.
Evidence of multiple operational affinities for d-glucose inside the human erythrocyte membrane
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1979
1. The Michaelis-Menten parameters of labelled D-glucose exit from human erythrocytes at 2°C into external solution containing 50 mM D-galactose were obtained. The Km is 3.4 + 0.4 mM, V 17.3 + 1.4 mmol • 1-1 cell water • min-1 for this infinite-trans exit procedure. 2. The kinetic parameters of equilibrium exchange of D-glucose at 2°C are Km= 25-+ 3.4 mM, V 30 + 4.1 mmol • 1-1 cell water • min-1. 3. The Km for net exit of D-glucose into solutions containing zero sugar is 15.8 + 1.7 mM, V 9.3 + 3.3 mmol • 1-~ cell water • min-1. 4. This experimental evidence corroborates the previous finding of Hankin, B.L., Lieb, W.R. and Stein, W.D. [(1972) Biochim. Biophys. Acta 255, 126-132] that there are sites with both high and low operational affinities for Dglucose at the inner surface of the human erythrocyte membrane. This result is inconsistent with current asymmetric carrier models of sugar transport.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1980
Mixed membrane vesicles prepared from cultured chick embryo fibroblasts possess a stereospecific D-glucose transport system, the properties of which are identical to those of the system in intact cells. Uptake of D-glucose proceeds without chemical alteration. The rate of stereospecific uptake of D-glucose into the mixed vesicles is 70% greater than that of the homogenate and uptake is directly proportional to membrane protein concentration. Stereospecific D-glucose uptake appears linear for 0.3 min, reaches a maximum at 2--5 min, and declines to zero by 5 h as L-glucose enters the vesicles. Uptake is osmotically sensitive and inhibited by cytochalasin B (K i = 0.13/~M) and the structural analogues of D-glucose : D-mannose, 2-deoxy-D-glucose, 3-O-methyl-Dglucose, D-galactose and maltose, but not by sucrose or L-glucose. Uphill counterflow can be demonstrated and the apparent activation energy displays a transition from 47.7 kcal/mol below 11°C to 18.1 kcal/mol above ll°C. Stereospecific uptake rates of mixed vesicles prepared from Rous sarcoma virustransformed cells are increased 30% over control values, and are increased 66% in vesicles derived from cells incubated for 24 h in glucose-free medium.
Human Erythrocyte Sugar Transport is Incompatible with Available Carrier Models
Biochemistry, 1996
GLUT1-mediated, passive D-glucose transport in human erythrocytes is asymmetric. V max and K m(app) for D-glucose uptake at 4°C are 10-fold lower than V max and K m(app) for D-glucose export. Transport asymmetry is not observed for GLUT1-mediated 3-O-methylglucose transport in rat, rabbit, and avian erythrocytes and rat adipocytes where V max for sugar uptake and exit are identical. This suggests that transport asymmetry is either an intrinsic catalytic property of human GLUT1 or that factors present in human erythrocytes affect GLUT1-mediated sugar transport. In the present study we assess human erythrocyte sugar transport asymmetry by direct measurement of sugar transport rates and by analysis of the effects of intra-and extracellular sugars on cytochalasin B binding to the sugar export site. We also perform internal consistency tests to determine whether the measured, steady-state 3-O-methylglucose transport properties of human erythrocytes agree with those expected of two hypothetical models for protein-mediated sugar transport. The simple-carrier hypothesis describes a transporter that alternately exposes sugar import and sugar export pathways. The fixed-site carrier hypothesis describes a sugar transporter that simultaneously exposes sugar import and sugar export pathways. Steady-state 3-Omethylglucose transport in human erythrocytes at 4°C is asymmetric. V max and K m(app) for sugar uptake are 10-fold lower than V max and K m(app) for sugar export. Phloretin-inhibitable cytochalasin B binding to intact red cells is unaffected by extracellular D-glucose but is competitively inhibited by intracellular D-glucose. This inhibition is reduced by 13% ( 4% when saturating extracellular D-glucose levels are also present. Assuming transport is mediated by a simple-carrier and that cytochalasin B and intracellular D-glucose binding sites are mutually exclusive, the cytochalasin B binding data are explained only if transport is almost symmetric (V max exit ) 1.4 V max entry). The cytochalasin B binding data are consistent with both symmetric and asymmetric fixed-site carriers. Analysis of 3-O-methylglucose, 2-deoxy-Dglucose, and D-glucose uptake in the presence of intracellular 3-O-methylglucose demonstrates significant divergence in experimental and theoretical transport behaviors. We conclude either that human erythrocyte sugar transport is mediated by a carrier mechanism that is fundamentally different from those considered previously or that human erythrocyte-specific factors prevent accurate determination of GLUT1-mediated sugar translocation across the cell membrane. We suggest that GLUT1-mediated sugar transport in all cells is an intrinsically symmetric process but that intracellular sugar complexation in human red cells prevents accurate determination of transport rates. Abstract published in AdVance ACS Abstracts, July 15, 1996. 1 Abbreviations used: CHO, Chinese hamster ovary; GLUT1, human erythrocyte glucose transport protein; 2DODG, 2-deoxy-D-glucose; 3OMG, 3-O-methylglucose; CCB, cytochalasin B; EDTA, ethylenediaminetetraacetic acid; HEPES, N-[2-hydroxyethyl]piperazine-N′-[2ethanesulfonic acid]; Tris-HCl, tris(hydroxymethyl)aminomethane. 2 S. A. Harrison, J. M. Buxton, A. Carruthers, unpublished findings.