Biomedical subjects

R J Zottola

Publications and source records attributed to R J Zottola.

3 recordsLinked to original sources

PubMed1995

Net sugar transport is a multistep process. Evidence for cytosolic sugar binding sites in erythrocytes.

Human erythrocyte net sugar transport is hypothesized to be rate-limited by reduced cytosolic diffusion of sugars and/or by reversible sugar association with intracellular macromolecules [Naftalin, R.J., Smith, P.M., & Roselaar, S.E. (1985) Biochim. Biophys. Acta 820, 235-249]. The present study examines these hypotheses. Protein-mediated 3-O-methylglucose uptake at 4 degrees C by human erythrocytes and by resealed, hypotonically lysed erythrocytes (ghosts) is inhibited by increasing solvent viscosity. Protein-mediated transport and transbilayer diffusion of the slowly transported substrate 6-NBD glucosamine are unaffected by increasing solvent viscosity. These findings suggest that protein-mediated 3-O-methylglucose transport is diffusion-limited in erythrocytes. More detailed analyses of red cell 3-O-methylglucose uptake (at 4 degrees C and at limiting extracellular sugar levels) reveal that net influx is a biexponential process characterized by rapid filling of a small compartment (C1 = 29 +/- 6% total cell volume; k1 = 7.4 +/- 1.7 min-1) and slow filling of a larger compartment (C2 = 71 +/- 6% total cell volume k2 = 0.56 +/- 0.11 min-1). Erythrocyte D-glucose net uptake at 4 degrees C is also a biphasic process. Transmembrane sugar leakage is a monoexponential process indicating that multicomponent, protein-mediated uptake does not result from sugar uptake by two cell populations of differing cellular volume. Sugar exit at limiting 3-O-methylglucose concentrations is described by single exponential kinetics. This demonstrates that multicomponent sugar uptake does not result from influx into two populations of cells with widely different sugar transporter content. We conclude that biexponential sugar uptake results from slow (relative to transport) exchange of sugars between serial, intracellular sugar compartments. Biexponential sugar uptake is observed under equilibrium exchange conditions (intracellular sugar concentration = extracellular sugar concentration) but only at 3-O-methylglucose concentrations of less than 1 mM. Above this sugar concentration, exchange uptake is a monoexponential process. Because diffusion rates are independent of diffusant concentration, this suggests that multicomponent uptake results from high-affinity sugar binding within the cell. The concentration of cytosolic binding sites (30 microM, Kd(app) = 400 microM) was estimated from the equilibrium cellular 3-O-methylglucose space. Biexponential net 3-O-methylglucose uptake is also observed in human erythrocyte ghosts, in control human K562 cells, and in K562 cells induced to synthesize hemoglobin by prolonged exposure to hemin. This demonstrates that neither membrane-bound nor free cytosolic hemoglobin forms the sugar-binding complex.(ABSTRACT TRUNCATED AT 400 WORDS)

3-O-Methylglucose↗

PubMed1995

Glucose transporter function is controlled by transporter oligomeric structure. A single, intramolecular disulfide promotes GLUT1 tetramerization.

The human erythrocyte glucose transporter is an allosteric complex of four GLUT1 proteins whose structure and substrate binding properties are stabilized by reductant-sensitive, noncovalent subunit interactions [Hebert, D. N., & Carruthers, A. (1992) J. Biol. Chem. 267, 23829-23838]. In the present study, we use biochemical and molecular approaches to isolate specific determinants of transporter oligomeric structure and transport function. When unfolded in denaturant, each subunit (GLUT1 protein) of the transporter complex exposes two sulfhydryl groups. Four additional thiol groups are accessible following subunit exposure to reductant. Assays of subunit disulfide bridge content suggest that two inaccessible sulfhydryl groups form an internal disulfide bridge. Differential alkylation/peptide mapping/N-terminal sequence analyses show that a GLUT1 carboxyl-terminal peptide (residues 232-492) contains three inaccessible sulfhydryl groups and that an N-terminal GLUT1 peptide (residues 147-261/299) contains two accessible thiols. The carboxyl-terminal peptide most likely contains the intramolecular disulfide bridge since neither its yield nor its electrophoretic mobility is altered by addition of reductant. Each GLUT1 cysteine was changed to serine by oligonucleotide-directed, in vitro mutagenesis. The resulting transport proteins were expressed in CHO cells and screened by immunofluorescence microscopy for their ability to expose tetrameric GLUT1-specific epitopes. Serine substitution at cysteine residues 133, 201, 207, and 429 does not inhibit exposure of tetrameric GLUT1-specific epitopes. Serine substitution at cysteines 347 or 421 prevents exposure of tetrameric GLUT1-specific epitopes. Hydrodynamic analysis of GLUT1/GLUT4 chimeras expressed in and subsequently solubilized from CHO cells indicates that GLUT1 residues 1-199 promote chimera dimerization and permit GLUT1/chimera heterotetramerization. This GLUT1 N-terminal domain is insufficient for chimera tetramerization which additionally requires GLUT1 residues 200-463. Extracellular reductants (dithiothreitol, beta-mercaptoethanol, or glutathione) reduce erythrocyte 3-O-methylglucose uptake by up to 15-fold. This noncompetitive inhibition of sugar uptake is reversed by the cell-impermeant, oxidized glutathione. Reductant is without effect on sugar exit from erythrocytes. Dithiothreitol doubles the cytochalasin B binding capacity of erythrocyte-resident glucose transporter, abolishes allosteric interactions between substrate binding sites on adjacent subunits, and occludes tetrameric GLUT1-specific GLUT1 epitopes in situ. CHO cell-resident GLUT1 structure and transport function are similarly affected by extracellular reductant. We conclude that each subunit of the glucose transporter contains an extracellular disulfide bridge (Cys347 and Cys421) that stabilizes transporter oligomeric structure and thereby accelerates transport function.

3-O-Methylglucose↗

PubMed1995

Rapid substrate translocation by the multisubunit, erythroid glucose transporter requires subunit associations but not cooperative ligand binding.

The human erythroid glucose transporter is a GLUT1 homotetramer whose structure and function are stabilized by noncovalent, cooperative subunit interactions. The present study demonstrates that exofacial tryptic digestion of GLUT1 abolishes cooperative interactions between substrate binding sites on adjacent subunits under circumstances where subunit associations and high catalytic turnover are maintained. Extracellular trypsin produces rapid, quantitative cleavage of the human red cell-resident sugar transport protein, GLUT1. One major carboxyl-terminal peptide of M(r)(app) 25,000 is detected by immunoblot analysis. Endofacial tryptic digestion of GLUT1 results in the complete loss of GLUT1 carboxyl-terminal structure. GLUT1-mediated erythrocyte sugar uptake, transport inhibition by cytochalasin B, and GLUT1 oligomeric structure are unaffected by exofacial GLUT1 proteolysis. In contrast, the cytochalasin B binding capacity of GLUT1 and the Kd(app) for cytochalasin B binding to the transporter are doubled following exofacial tryptic digestion of GLUT1. Photoaffinity labeling experiments show that increased cytochalasin B binding results from increased ligand binding to the 25 kDa carboxyl-terminal GLUT1 peptide. Proteolysis abolishes allosteric interactions between sugar import (maltose binding) and sugar export (cytochalasin B binding) sites that normally exist on adjacent subunits within the transporter complex, but interact with negative cooperativity. Following exofacial proteolysis, these sites become mutually exclusive. Dithiothreitol disrupts GLUT1 quaternary structure, inhibits 3-O-methylglucose transport, and abolishes cooperative interactions between sugar import and export sites in control cells. Studies with reconstituted purified GLUT1 confirm that the action of trypsin on cytochalasin B binding is direct, show that proteolysis increases the apparent affinity of the sugar efflux site for transported sugars, and suggest that the membrane bilayer stabilizes GLUT1 noncovalent structure and catalytic function following GLUT1 proteolysis. Collectively, these findings demonstrate that GLUT1 does not require an intact polypeptide backbone for catalytic function. They show that the multisite sugar transporter mechanism is converted to a simple ping-pong carrier mechanism following exofacial GLUT1 proteolysis. They reveal that subunit cooperativity can be lost under circumstances where cohesive structural interactions between transporter subunits are maintained. They also refute the hypothesis [Hebert, D. N., & Carruthers, A. (1992) J. Biol. Chem. 267, 23829-23838] that rapid substrate translocation by the multisubunit erythroid glucose transporter requires cooperative interactions between subunit ligand binding sites.

3-O-Methylglucose↗