Structural basis of human erythrocyte glucose transporter function in reconstituted system. Hydrogen exchange.
Hydrogen exchange kinetic behavior of human erythrocyte glucose transporter protein in vesicles was studied in the absence and in the presence of D-glucose or a well known inhibitor, cytochalasin B. This is to detect a proposed channel of water penetrating into the protein through which the sugar molecule passes and to monitor any conformational changes induced by the substrate or inhibitor. Analyses of the kinetic data revealed several classes of hydrogens which exchange with readily distinguishable rates. Of 660 hydrogens detected per transporter, approximately 30% exchanged with rates generally characterized as those of free amide hydrogens indicating they are interfaced to solvent water. Since the transporter is known to be embedded deep in the hydrophobic area of the membrane with minimum exposure to the outside of the membrane lipid bilayer, a significant portion of these free amide hydrogens must be at the purported channel rather than outside of the membrane. D-Glucose and cytochalasin B affected the exchange kinetics of these presumably channel-associated free amide hydrogens rather differently. D-Glucose reduced the apparent rate constants, but not the total number. Cytochalasin B on the other hand reduced the total number to one-half without significantly changing the apparent rate constants. The remaining 70% of the labeled hydrogens exchanged with much slower rates which vary 10-10,000-fold, indicating that they are internally structured peptide amide and side chain hydrogens. Both D-glucose and cytochalasin B further reduced the rates of these hydrogens, indicating a global stabilization of the protein structure.
D003571:Cytochalasin B / Q000494:pharmacology*; D003903:Deuterium; D004910:Erythrocyte Membrane / Q000032:analysis*; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D006859:Hydrogen / Q000097:blood*; D007700:Kinetics; D009004:Monosaccharide Transport Proteins / Q000097:blood*; D011487:Protein Conformation; D013055:Spectrophotometry, Infrared
D009004:Monosaccharide Transport Proteins; D003571:Cytochalasin B; D006859:Hydrogen; D003903:Deuterium; D005947:Glucose