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Photolabelling of the hexose transporter at external and internal sites: fragmentation patterns and evidence for a
Abstract:
The human erythrocyte sugar transporter has been labelled at its internal site with cytochalasin B and at its outside site by the azidosalicoyl derivative of bis(D-mannose) (ASA-BMPA). The cleavage of the transporter by various proteinases has been studied. Chymotrypsin, subtilisin and V8 proteinase give parallel fragmentation patterns for the two labels down to fragments as small as 7 kDa. Thus the binding sites for the two labels can only be separated by a small span of protein. 2-Nitro-5-thiocyanobenzoic acid (NTCB) cleaves at cysteines to give a 15 kDa fragment from the two labels. N-Bromosuccinimide (a reagent which preferentially cleaves at tryptophan residues) has revealed differences in fragmentation of the transporter labelled with either cytochalasin B or with ASA-BMPA. A major cleavage site is proposed to occur at tryptophan 186 which leaves a C-terminal fragment containing both labels. A tryptophan cleavage at residue 388 divides the cytochalasin B site and the ASA-BMPA site. A further tryptophan cleavage gives a cytochalasin B labelled 3 kDa fragment probably from residues 388-412. This gives an assignment of the cytochalasin B site as the inside of the hydrophobic span H 10. Since the ASA-BMPA site is probably only 7 kDa from residue 388 and is on the same 15 kDa NTCB fragment as cytochalasin B we assign this to the outside of hydrophobic span H 9. Thermolysin only cleaves the transporter labelled with cytochalasin B and not with ASA-BMPA. A 18 kDa cytochalasin B labelled fragment is formed. This is indicative of a change in conformation of the transporter when an outside ligand is bound such that the inside of the hydrogen bonding transmembrane segments H 7 and H 8 (and containing the proposed thermolysin cleavage site) are withdrawn from the cytosolic surface. Thus it appears that the core of the transporter (including the external and internal sites plus the transmembrane channel) is located between segments H 7 and H 10.