Cytochalasin B as a probe of protein structure and substrate recognition by the galactose/H+ transporter of Escherichia coli.
Cytochalasin B is a potent inhibitor of mammalian passive glucose transporters. The recent demonstration of sequence similarities between these proteins and several bacterial proton-linked sugar transporters suggested that cytochalasin B might be a useful tool for investigation of the galactose/H+ symport protein (GalP) of Escherichia coli. Equilibrium binding studies using membranes from a GalP-constitutive (GalPc) strain of E. coli revealed a single set of high affinity binding sites for cytochalasin B with a Kd of 0.8-2.2 microM. Binding was inhibited by D-glucose, but not by L-glucose. UV irradiation of the membranes in the presence of [4-3H]cytochalasin B photolabeled principally a protein of apparent Mr 38,000, corresponding to the GalP protein. Labeling was inhibited by greater than 80% in the presence of 500 mM D-glucose or D-galactose, the major substrates of the GalP system. The extent of inhibition of photolabeling by different sugars and sugar analogues showed that the substrate specificity of GalP closely resembles that of the mammalian passive glucose transporters. Structural similarity to the latter was revealed by tryptic digestion of [4-3H]cytochalasin B-photolabeled GalP, which yielded a radiolabeled fragment of apparent Mr 17,000-19,000, similar to that previously reported for the human erythrocyte glucose transporter.
Cairns
M T
MT
Department of Biochemistry, Royal Free Hospital School of Medicine, University of London, United Kingdom.
McDonald
T P
TP
Horne
P
P
Henderson
P J
PJ
Baldwin
S A
SA
D001665:Binding Sites; D001693:Biological Transport, Active; D050260:Carbohydrate Metabolism; D003571:Cytochalasin B / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D005690:Galactose / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D011522:Protons; D014357:Trypsin / Q000378:metabolism
D009004:Monosaccharide Transport Proteins; D011522:Protons; D003571:Cytochalasin B; D014357:Trypsin; D005947:Glucose; D005690:Galactose