Photolabeling of erythrocyte and adipocyte hexose transporters using a benzophenone derivative of bis(D-mannose).
The benzophenone derivative of 1,3-bis(D-mannos-4-yloxy)-2-propylamine (BB-BMPA) has been tested as an exofacial photoaffinity label for the sugar transport systems of human erythrocytes and rat adipocytes. The half-maximal inhibition constants for the reagent are 971 microM in erythrocytes and 536 microM in basal and 254 microM in insulin-treated adipocytes. The photolabelling of erythrocyte membranes is very specific for the 50 kDa transporter peptide and is completely displaced by D-glucose. The exofacial photoaffinity labelling of adipocytes also shows labelling of a 50 kDa transporter peptide, which is displaced by cytochalasin B, but extensive nonspecific labelling of a 75 kDa plasma membrane peptide occurs. The transporter is labelled in insulin-treated cells but not in basal cells which indicates that this in situ labelling technique selectively reveals only those transporters that visit and are active in the plasma membrane during the labelling period. This also indicates that in basal cells transporters do not turn over rapidly. Subcellular redistribution of transporters after the labelling period has been studied. Following incubation and washing at 37 degrees C in the presence of insulin, 30% of the transporters photolabelled at the plasma membrane are internalised and are found in the light microsome fraction of the cell. The proportion of transporter that is observed to be internalised is much greater than can be accounted for by a contamination of the light microsome fraction by plasma membrane. The labelled 50 kDa transporter peptide in the light microsomes is enriched when compared with the carry-over of the 75 kDa nonspecifically labelled plasma membrane peptide. Thus we have obtained direct evidence for transporter translocation.
Holman
G D
GD
Department of Biochemistry, University of Bath, U.K.
Karim
A R
AR
Karim
B
B
D000273:Adipose Tissue / Q000378:metabolism*; D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D003571:Cytochalasin B / Q000378:metabolism; D004187:Disaccharides / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D010777:Photochemistry; D051381:Rats
D000345:Affinity Labels; D004187:Disaccharides; D009004:Monosaccharide Transport Proteins; C058174:2-N-(4-benzoyl)benzoyl-1,3-bis(mannos-4-yloxy)-2-propylamine; D003571:Cytochalasin B