Evidence for two asymmetric conformational states in the human erythrocyte sugar-transport system

Abstract

6-O-methyl-, 6-O-propyl-, 6-O-pentyl- and 6-O-benzyl-D-galactose, and 6-O-methyl-, 6-O-propyl- and 6-O-pentyl-D-glucose inhibit the glucose-transport system of the human erythrocyte when added to the external medium. Penetration of 6-O-methyl-D-galactose is inhibited by D-glucose, suggesting that it is transported by the glucose-transport system, but the longer-chain 6-O-alkyl-D-galactoses penetrate by a slower D-glucose-insensitive route at rates proportional to their olive oil/water partition coefficients. 6-O-n-Propyl-D-glucose and 6-O-n-propyl-D-galactose do not significantly inhibit L-sorbose entry or D-glucose exit when present only on the inside of the cells whereas propyl-beta-D-glucopyranoside, which also penetrates the membrane slowly by a glucose-insensitive route, only inhibits L-sorbose entry or D-glucose exit when present inside the cells, and not when on the outside. The 6-O-alkyl-D-galactoses, like the other nontransported C-4 and C-6 derivatives, maltose and 4,6-O-ethylidene-D-glucose, protect against fluorodinitrobenzene inactivation, whereas propyl beta-D-glucopyranoside stimulates the inactivation. Of the transported sugars tested, those modified at C-1, C-2 and C-3 enhance fluorodinitrobenzene inactivation, where those modified at C-4 and C-6 do not, but are inert or protect against inactivation. An asymmetric mechanism is proposed with two conformational states in which the sugar binds to the transport system so that C-4 and C-6 are in contact with the solvent on the outside and C-1 is in contact with the solvent on the inside of the cell. It is suggested that fluorodinitrobenzene reacts with the form of the transport system that binds sugars at the inner side of the membrane. An Appendix describes the theoretical basis of the experimental methods used for the determination of kinetic constants for non-permeating inhibitors.

Substrate Recognition at the Cytoplasmic and Extracellular Binding Site of the Lactose Transport Protein of Streptococcus thermophilus

“…The hydrophobic surface incorporating these hydrogen atoms would be a candidate for interaction with aromatic residues in the binding site. In contrast, bound [1][2][3][4][5][6][7][8][9][10][11][12][13] C]D-galactose observed with cross-polarization magic-angle spinning NMR showed no significant difference in chemical shift compared with galactose in free solution, which is indicative of a comparable chemical environment (24). This would be consistent with C-1 being remote from the hydrophobic surface, as viewed in Fig.…”

“…We speculate that the C-2-OH and C-6-OH contribute highly to the affinity for galactose at the cytoplasmic binding site by forming hydrogen bonds with the protein, which does not take place when galactose is bound at the extracellular binding site (Table IV). Differences in architecture of the cytoplasmic and extracellular binding site surrounding the substrate have also been reported for Glut 1 and GalP (4,13). In these cases the differences do not represent differences in interactions of the binding sites with the hydroxyl groups, but rather differences in interactions with bulky substituents.…”

Analysis of Transmembrane Segment 8 of the GLUT1 Glucose Transporter by Cysteine-scanning Mutagenesis and Substituted Cysteine Accessibility

“…Remarkably, the structures suggest analogous mechanisms for cotransport and antiport and, by inference, simple uniport involving the tilting of helices such that substrate binding sites are alternately exposed to either the cytoplasm or exoplasm. An alternating conformation mechanism of this type was originally postulated by Vidaver (28) nearly four decades ago based purely on kinetic considerations, and inhibitor (29) and spectroscopic studies (30,31) on the red cell glucose transporter have strongly supported such a mechanism.…”

Will the original glucose transporter isoform please stand up!

“…Competitive inhibition studies by Barnett et al (7) suggest that the hydroxyl (OH) groups at C1 and C3 of D-glucose serve as hydrogen bond acceptors when D-glucose is seated in the GLUT1 sugar uptake site. C4 may form a hydrogen bond with GLUT1 because the C4 epimer of D-glucose, D-galactose, has 10-fold lower affinity for GLUT1 than D-glucose.…”