Cell-surface recognition of biotinylated membrane proteins requires very long spacer arms: an example from glucose-transporter probes.
Glucose transporters (GLUTs) can be photoaffinity labelled by (diazirinetrifluoroethyl)benzoyl-substituted glucose derivatives and the adduct can be recognised, after detergent solubilisation of membranes, by using streptavidin-based detection systems. However, in intact cells recognition of photolabelled GLUTs by avidin and anti-biotin antibodies only occurs if the bridge between the photoreactive and the biotin moieties has a minimum of 60--70 spacer atoms. We show that a suitably long bridge can be synthesised with a combination of polyethylene glycol and tartarate groups and that introduction of these spacers generates hydrophilic products that can be cleaved with periodate. Introduction of the very long spacers does not appreciably reduce the affinity of interaction of the probes with the transport system.
Hashimoto
M
M
Department of Biology and Biochemistry, University of Bath, Bath BA27AY, UK.
Yang
J
J
Holman
G D
GD
D017667:Adipocytes / Q000187:drug effects / Q000201:enzymology; D000818:Animals; D001360:Avidin / Q000737:chemistry; D001710:Biotin / Q000737:chemistry*; D004912:Erythrocytes / Q000378:metabolism; D051275:Glucose Transporter Type 4; D006801:Humans; D007120:Immunochemistry; D008565:Membrane Proteins / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D008968:Molecular Conformation; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D009124:Muscle Proteins; D019920:Photoaffinity Labels; D051381:Rats; D011956:Receptors, Cell Surface / Q000737:chemistry*
D051275:Glucose Transporter Type 4; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D019920:Photoaffinity Labels; D011956:Receptors, Cell Surface; C495692:SLC2A4 protein, human; C495694:Slc2a4 protein, rat; D001360:Avidin; D001710:Biotin