Photoaffinity labeling of the stereospecific D-glucose transport system with cytochalasin B.
A method has been developed for photoaffinity labeling components of the hexose transport system with [3H]cytochalasin B. We have demonstrated that UV photoirradiation of intact human erythrocytes or ghost membranes with 0.5 microM [3H]cytochalasin B yielded a broad peak with an Mr of 44,000-70,000. Labeling that was insensitive to the presence of 0.5 M D-sorbitol was substantially inhibited by 0.5 M D-glucose. Approximately 80% of labeling in the region of Mr = 49,000-70,000 was inhibited by the presence of 0.5 M D-glucose, whereas labeling of the 44,000- to 49,000-dalton region was inhibited only 30%. Somewhat different results were obtained from photoaffinity labeling of plasma membranes from chicken embryo fibroblasts (CEF). The [3H]cytochalasin B-labeling patterns had two relatively sharp and discrete peaks at 46,000 and 52,000 daltons. Comparison of plasma membranes from glucose-fed and starved CEF revealed that the total D-glucose-sensitive labeling increased approximately 12-fold in the starved cell membranes. Labeling of the 52,000-dalton polypeptide was more sensitive than that of the 46,000-dalton polypeptide to inhibition by the presence of D-glucose. These results indicate that [3H]cytochalasin B photoaffinity labeling has wide applicability for identifying and covalently binding components of the facilitated hexose transport system.
D000345:Affinity Labels; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism / Q000528:radiation effects; D002478:Cells, Cultured; D002642:Chick Embryo; D003571:Cytochalasin B; D004910:Erythrocyte Membrane / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D006801:Humans; D009004:Monosaccharide Transport Proteins; D010777:Photochemistry; D014316:Tritium; D014466:Ultraviolet Rays
D000345:Affinity Labels; D002352:Carrier Proteins; D009004:Monosaccharide Transport Proteins; D014316:Tritium; D003571:Cytochalasin B; D005947:Glucose