Immobilized proteoliposome affinity chromatography for quantitative analysis of specific interactions between solutes and membrane proteins. Interaction of cytochalasin B and D-glucose with the glucose transporter Glut1.
An affinity gel bed was prepared by reconstitution of a transmembrane protein, the human red cell glucose transporter (Glut1), followed by steric immobilization of the proteoliposomes in small and rigid gel beads by freeze-thawing. The specific interactions between the reconstituted Glut1, the transport inhibitor cytochalasin B (CB), and the transported solute D-glucose were analyzed by isocratic chromatography of CB on the Glut1-proteoliposome gel bed. Specific retardation of CB which decreased upon inclusion of the competitor D-glucose in the eluent was observed on-line. The equilibrium constants for CB and D-glucose interaction with Glut1 (Kd 1.5 x 10(-7) M and 67 mM, respectively) obtained by use of equations derived for the affinity chromatographic analysis were consistent with values obtained by others by conventional methods. Effects of liposome composition, pH, and time on the CB binding activity of Glut1 were studied. Reconstitution of a membrane protein into a lipid environment and steric immobilization of the proteoliposomes favor retention of the protein activity. Immobilized proteoliposome affinity chromatography (IPAC) is a novel, powerful method for analysis of interactions between membrane proteins and solutes.
Yang
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Department of Biochemistry, Uppsala University, Sweden.
Lundahl
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P
D002846:Chromatography, Affinity / Q000379:methods*; D003571:Cytochalasin B / Q000302:isolation & purification*; D004910:Erythrocyte Membrane / Q000378:metabolism; D005947:Glucose; D051272:Glucose Transporter Type 1; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D008081:Liposomes; D008433:Mathematics; D008563:Membrane Lipids / Q000097:blood; D008565:Membrane Proteins / Q000097:blood / Q000378:metabolism; D008962:Models, Theoretical; D009004:Monosaccharide Transport Proteins; D011510:Proteolipids; D012996:Solutions
D051272:Glucose Transporter Type 1; D007202:Indicators and Reagents; D008081:Liposomes; D008563:Membrane Lipids; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D011510:Proteolipids; C495677:SLC2A1 protein, human; D012996:Solutions; C020485:proteoliposomes; D003571:Cytochalasin B; D005947:Glucose