Phloretinyl-3′-benzylazide: A high affinity probe for the sugar transporter in human erythrocytes I. Hexose transport inhibition and photolabeling of mutarotase

Fannin, Franklin F.; Evans, James O.; Gibbs, E. Michael; Diedrich, Donald F.

doi:10.1016/0005-2736(81)90406-5

Cited by 14 publications

(16 citation statements)

References 42 publications

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“…Later studies demonstrated that it actually enters red cells rapidly and is strongly adsorbed both to the cell membrane and to hemoglobin [9,14]. Hence phloretin could conceivably add to the carrier both inside and outside, and several authors have suggested that it does so.…”

Section: The Asymmetry Of the Glucose Carrier And Phloretin Bindingmentioning

confidence: 97%

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Asymmetrical binding of phloretin to the glucose transport system of human erythrocytes

Krupka

1985

J. Membrain Biol.

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The sidedness of phloretin binding to the glucose carrier has been determined by comparing the type of inhibition produced in zero trans entry and zero trans exit experiments. Initial rates of zero trans entry were measured by the method of R.D. Taverna and R.G. Langdon (Biochim. Biophys. Acta 298:412-421, 1973), which involves pink ghosts loaded with glucose oxidase; this obviates the problem of rapid substrate accumulation inside the cells. With phloretin equilibrated across the membrane, the inhibition of entry was competitive, and the inhibition of exit noncompetitive. The experimental procedures were validated by showing that the inhibition by cytochalasin B, known to bind inside but not outside, was noncompetitive in entry and competitive in exit, as predicted. It was also demonstrated that even after pre-incubation of the cells with a relatively high concentration of phloretin, the phloretin adsorbed in the membrane did not significantly alter the rate of carrier reorientation. The results show that the outward-facing form of the glucose carrier, but not the inward-facing form, bears a phloretin binding site; thus phloretin, as well as cytochalasin B, is bound asymmetrically, phloretin outside and cytochalasin B inside.

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Section: The Asymmetry Of the Glucose Carrier And Phloretin Bindingmentioning

confidence: 97%

“…Phloretin resembles cytochalasin B in having an exceptionally high affinity for the carrier and in binding, reversibly, in competition with the substrate [3,9,17]--though neither inhibitor is a structural analog of glucose; but the question of where phloretin binds has not yet been settled.…”

Section: The Asymmetry Of the Glucose Carrier And Phloretin Bindingmentioning

confidence: 98%

Asymmetrical binding of phloretin to the glucose transport system of human erythrocytes

Krupka

1985

J. Membrain Biol.

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“…Its transport is to be expected, in view of the wide range of organic anions accepted by the system. After rapidly equilibrating across the cell membrane by simple diffusion (Jennings & Solomon, 1976;Fannin et al, 1981), phloretin could hypothetically add to both the inner and outer carrier sites. By adding to the inner, it would reduce the rate of outward C1-translocation in both exchange and net exit experiments.…”

Section: Gomentioning

confidence: 99%

Role of substrate binding forces in exchange-only transport systems: II. Implications for the mechanism of the anion exchanger of red cells

1989

J. Membrain Biol.

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The transition-state theory of exchange-only membrane transport is applied to experimental results in the literature on the anion exchanger of red cells. Two central features of the system are in accord with the theory: (i) forming the transition state in translocation involves a carrier conformational change; (ii) substrate specificity is expressed in transport rates rather than affinities. The expression of specificity is consistent with other evidence for a conformational intermediate (not the transition state) formed in the translocation of all substrates. The theory, in conjunction with concepts derived from the chemistry of macrocyclic ion inclusion complexes, prescribes certain essential properties in the transport site. Separate subsites are required for the preferred substrates, Cl- and HCO3-, to account for tight binding in the transition state (Kdiss congruent to 1 microM). Further, the following mechanism is suggested. A substrate anion initially forms a loose surface complex at one subsite, but in the transition state the subsites converge to form an inclusion complex in which the binding forces are greatly increased through a chelation effect. The conformational change at the substrate site, which is driven by the mounting forces of binding, sets in train a wider conformational change that converts the carrier from an immobile to a mobile form. Though simple, this composite-site mechanism explains many unusual features of the system. It accounts for substrate inhibition, partially noncompetitive inhibition of one substrate by another, and "tunneling," which is net transport under conditions where exchange should prevail, according to other models. All three types of behavior result from the formation of a ternary complex in which substrate anions are bound at both subsites. The mechanism also accounts for the enormous range of substrate structures accepted by the system, for the complex inhibition by the organic sulfate NAP-taurine, and for the involvement of several cationic side chains and two different protein domains in the transport site.

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“…After standing for an additional 2 days at 4"C, the mixture was poured into a column and unreacted PBA was washed from the beads with 50% ethylene glycol-water. The recovery of uncoupled PBA in the column eluate was analyzed spectrophotometrically (t332 = 21.0 x lo3 M-' cm-' in 0.025 M Na2B407, pH 9.3 [9]). …”

Section: Preparation Of the Affinity Media: Pba Sepharosementioning

confidence: 99%

“…1) and coupled to commercially available agarose derivatives. PBA is even more potent than phloretin as a strictly competitive inhibitor of glucose influx in human erythrocytes with almost lo4 times the affinity for the carrier site as glucose [9]. The benzylamine group on phloretin's A ring allows the easy attachment of the compound to insoluble polymers while preserving the features of the ligand required for transport inhibitory properties.…”

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confidence: 99%

Solubilization, reconstitution, and attempted affinity chromatography of the sugar transporter of the erythrocyte membrane

Weber

Warden

Semenza

et al. 1985

J of Cellular Biochemistry

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Reconstitution of the sugar transport system of human erythrocytes into artificial liposomes was achieved by freezing, thawing, and sonicating preformed phospholipid vesicles in the presence of intact ghosts, protein-depleted ghosts, or detergent-treated ghosts. D-glucose equilibrium exchange activities and affinity constants in the range of the reported erythrocyte values were reached in the best experiments. Whereas the extraction of peripheral membrane proteins did not depress the transport function crucially after reconstituting these protein-depleted ghosts, the selective solubilization of integral membrane proteins by a variety of nonionic detergents resulted in an uncontrollable, continuously increasing inactivation of the carrier. However, Emulphogene BC-720 extracts could be prepared in which the glucose transporter retained activity for days at 4 degrees C. These extracts were applied to affinity chromatography matrices of phloretin-Agarose, prepared by coupling phloretinyl-3'-benzylamine (PBA) to CH-Sepharose 4B and to Affigel 202. Although the solubilized sugar transporter appeared to be selectively adsorbed to both PBA matrices, it could not be eluted by specific counter ligands or gentle eluants in a biologically active form. However, chaotropic agents could be used to elute intrinsic proteins, including bands 3 and 4.5, from the Affigel affinity medium.

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Phloretinyl-3′-benzylazide: A high affinity probe for the sugar transporter in human erythrocytes I. Hexose transport inhibition and photolabeling of mutarotase

Cited by 14 publications

References 42 publications

Asymmetrical binding of phloretin to the glucose transport system of human erythrocytes

Asymmetrical binding of phloretin to the glucose transport system of human erythrocytes

Role of substrate binding forces in exchange-only transport systems: II. Implications for the mechanism of the anion exchanger of red cells

Solubilization, reconstitution, and attempted affinity chromatography of the sugar transporter of the erythrocyte membrane

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