Reevaluation of use of retardation chromatography to demonstrate selective monosaccharide “binding” by erythrocyte membranes

LeFevre, Paul G.; Masiak, Stanley J.

doi:10.1007/bf01868025

Cited by 16 publications

(8 citation statements)

References 12 publications

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“…To be sure, there is no definite knowledge of the actual mechanism of transport in this cell type. Attempts at isolating a membrane protein that would show affinity for glucose (Bobinski & Stein, 1966) have failed, perhaps because of the very low affinity of the membrane binding sites for glucose but perhaps also because there are no isolatable binding components in the erythrocyte membrane (see also LeFevre & Masiak, 1970). This has led to the suggestion of non-carrier, lattice-type models (Lieb & Stein, 1970;Naftalin, 1970) which can do without the classical carrier concept, and still account for phenomena like counter-transport and competitive acceleration.…”

Section: Discussionmentioning

confidence: 99%

Effect of phloretin on monosaccharide transport in erythrocyte ghosts

Beneš¹,

Kolínská²

1972

J. Membrain Biol.

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Summary. 3H-labelled phloretin was shown to be bound reversibly by human erythrocyte and ghost membranes but not to penetrate across them in either direction. Kinetic parameters of D-xylose and D-galactose transport in intact cells and in ghosts, as well as the inhibition by phloretin of these transports were found to be in fair agreement. By enclosing phloretin in ghosts, its inhibition of monosaccharide transport was found to be symmetrical and thus an equivalence of the outer and the innner membrane sides of the human erythrocyte was demonstrated.

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Section: Discussionmentioning

confidence: 99%

Effect of phloretin on monosaccharide transport in erythrocyte ghosts

Beneš¹,

Kolínská²

1972

J. Membrain Biol.

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“…Other attempts to isolate the essential component(s) of the sugar transport system based on glucose binding or reconstitution were not successful (2,(10)(11)(12)(13)(14), see also ref. 15). How-ever, other membrane transport systems have been reconstituted in liposomes by several methods (16)(17)(18)(19)(20)(21)(22).…”

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confidence: 99%

Reconstitution of D-glucose transport catalyzed by a protein fraction from human erythrocytes in sonicated liposomes.

Kasahara¹,

Hinkle²

1976

Proc. Natl. Acad. Sci. U.S.A.

159

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A protein fraction was obtained from human erythrocyte ghosts by solubilization with Triton X-100 or octylglucoside. Triton X-100 was removed from the protein by Bio-Beads SM-2 and octylglucoside, by diafiltration. The solubilized protein fraction catalyzed D-glucose uptake when reconstituted in sonicated liposomes. The min at 370, and sedimented at 78,000 X g for 30 min. The pellet was washed once with 100 ml of 0.1 mM EDTA, treated with 100 ml of 0.5 M NaCl, 5 mM Tris-H2SO4 (pH 7.4) for 20 min at 40, washed once with 50 ml of the same salt solution and twice with 50 ml of 10 mM Tris-H2SO4 (pH 7.4), and stored at -70°(5 mg of protein per ml in 10 mM Tris, H2SO4). We confirmed the previous observation that EDTA and NaCl treatment removed several proteins from the ghosts and converted the ghosts into small vesicles (25). Solubilization with detergents Solubilization with Triton X-100. Triton X-100 was treated with SnCl2 to remove peroxides (26,27). Most of the peroxides (more than 85%) were removed, as estimated by the ferrous thiocyanate method (28). Sodium-chloride-treated vesicles (40 mg of protein in 20 ml) were incubated with 0.5% of purified Triton X-100 in 10 mM Tris-H2SO4 (pH 7.4) for 20 min at 4°. After centrifugation at 100,000 X g for 60 min, the supernatant was treated with Bio-Beads SM-2 overnight (6 g wet weight of beads per 20 ml) at 40 (29), divided into small fractions, and stored at -70°.Solubilization with Octylglucoside. Sodium-chloridetreated vesicles (14 mg of protein in 7 ml) were incubated with 30 mM octylglucoside in 10 mM Tris-H2SO4 (pH 7.4) for 30 min at 4°. After centrifugation at 100,000 X g for 60 min, the supernatant was subjected to diafiltration (XM-50; cut-off molecular weight, 50,000) against 10 mM TrisH2SO4 (pH 7.4) for 6 hr to overnight at 40. The protein fraction was divided into small fractions and stored at -70°. Reconstitution of D-glucose uptake system Acetone-washed soybean phospholipids (16) in a deoxygenated buffer (150 mM KC1, 10 mM Tris.HCl, pH 7.4) were sonicated in a test tube for 15-30 min at 20-400 with a sonicator (80 W, 80 kHz, Generator model G80-80-1, Tank model T80-80-1-R$, Laboratory Supplies, Hicksville, N.Y.) at 25-40 mg dry weight of lipid per ml. The solubilized membrane fraction was added to the sonicated liposomes, 15-20 mg/ml of phospholipids and 0.5-1.0 mg/ml of protein (total volume, 0.5-1.5 ml). The mixture was flushed with N2 gas and sonicated further for 1-3 min at 30-35' (30). The reconstitution was not specific for the soybean phospholipids; a mixture of egg phosphatidylcholine and soybean phosphatidylethanolamine could be used in place of the soybean phospholipids. Uptake of radioactive sugars A portion (85-120 gl)

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“…The carrier molecule is generally assumed to be a specific membrane protein or lipoprotein and although considerable effort has gone into the isolation of the carrier molecule, the results to date have been uniformly unsuccessful (LeFevre & Masiak, 1970;Masiak & LeFevre, 1972).…”

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confidence: 99%

Interaction of sugar acetals with the human erythrocyte glucose transport system

Novak

LeFevre

1974

J. Membrain Biol.

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Summary. All of the eleven tested bi-and tricyclic sugar acetals were found to be competitive inhibitors of glucose exit from human red cells. The highest-affinity agents were those locked into a CI conformation by fusion with a m-dioxane ring attached to a phenyl group (methyl-4,6-O-benzylidene-o-glucopyranoside, K i = 0.73 mM; 4,6-O-benzylidene-o-glucopyranose, /s mM at 23 ~ All of the isopropylidene acetals had less affinity than D-glucose (Sen-Widdas Kin=2.5 raM) for the transport system. Sugars in which two or more hydroxyl groups were cross-linked by isopropylidene groups apparently penetrated the red cell membrane by simple diffusion, as shown by a lack of (1) saturation in the entry process, (2) competition for entry with other acetals or D-glucose, and (3) inhibition by phloretin or Hg 2+ on entry rates.

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Reevaluation of use of retardation chromatography to demonstrate selective monosaccharide “binding” by erythrocyte membranes

Cited by 16 publications

References 12 publications

Effect of phloretin on monosaccharide transport in erythrocyte ghosts

Effect of phloretin on monosaccharide transport in erythrocyte ghosts

Reconstitution of D-glucose transport catalyzed by a protein fraction from human erythrocytes in sonicated liposomes.

Interaction of sugar acetals with the human erythrocyte glucose transport system

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