Inhibition of hexose transport and labelling of the hexose carrier in human erythrocytes by an impermeant maleimide derivative of maltose.
Maltose-maleimide was synthesized as a potential affinity label for the facilitative hexose carrier with selectivity for exofacial sulphydryl groups. This reagent, although probably a mixture of isomers, did not significantly penetrate the plasma membrane of human erythrocytes at concentrations below 5 mM at 37 degrees C. When allowed to react to completion, it irreversibly inhibited the uptake of 3-O-methylglucose, with a half-maximal response at about 1.5-2.0 mM-reagent. The rate of transport inactivation was a saturable function of the maltose-maleimide concentration. Studies of reaction kinetics and effects of known transport inhibitors demonstrated that irreversible reaction occurred on the exofacial outward-facing carrier, although not at a site involved in substrate binding. Reaction of intact erythrocytes with [14C]maltose-maleimide resulted in labelling of a broad band 4.5 protein of Mr (average) 45,000-66,000 in electrophoretic gels. This protein was very likely the hexose carrier, since its labelling was inhibited by cytochalasin B. Exofacial band 4.5 labelling was stoichiometric with respect to transport inhibition, yielding an estimated 300,000 carriers/cell. These results suggest that the exofacial sulphydryl which reacts with maltose-maleimide is distinct from the substrate binding site on the hexose carrier, but that it confers substantial labelling selectivity to impermeant maleimides. Additionally, the high efficiency of carrier labelling obtained with maltose-maleimide is useful in quantifying numbers of carriers in whole cells.
May
J M
JM
Diabetes Research and Training Center, Vanderbilt University School of Medicine, Nashville, TN 37232.
D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000097:blood*; D003572:Cytochalasins / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology; D006601:Hexoses / Q000097:blood*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008301:Maleimides / Q000097:blood*; D008320:Maltose / Q000031:analogs & derivatives* / Q000097:blood; D013696:Temperature
D002352:Carrier Proteins; D003572:Cytochalasins; D006601:Hexoses; D008301:Maleimides; C057394:maltose-maleimide; D008320:Maltose; D005947:Glucose