Cytochalasin B as a probe of protein structure and substrate recognition by the galactose/H+ transporter of Escherichia coli

• Published in: The Journal of biological chemistry 1991-05, Vol.266 (13), p.8176-8183
• Main Authors: CAIRNS, M. T, MCDONALD, T. P, HORNE, P, HENDERSON, P. J. F, BALDWIN, S. A
• Format: Article
• Language: English
• Subjects: 550201 - Biochemistry- Tracer Techniques; 560120 - Radiation Effects on Biochemicals, Cells, & Tissue Culture; ALDEHYDES; BACTERIA; BARYONS; BASIC BIOLOGICAL SCIENCES; Binding Sites; BIOCHEMICAL REACTION KINETICS; Biological and medical sciences; BIOLOGICAL FUNCTIONS; Biological Transport, Active; Carbohydrate Metabolism; CARBOHYDRATES; CELL CONSTITUENTS; CELL MEMBRANES; Cell physiology; CHEMICAL RADIATION EFFECTS; Cytochalasin B - metabolism; ELEMENTARY PARTICLES; ENZYMES; ESCHERICHIA COLI; Escherichia coli - metabolism; FERMIONS; Fundamental and applied biological sciences. Psychology; GALACTOSE; Galactose - metabolism; GLUCOSE; Glucose - metabolism; HADRONS; HEXOSES; HYDROGEN COMPOUNDS; HYDROLASES; ISOTOPE APPLICATIONS; KINETICS; Membrane and intracellular transports; MEMBRANE PROTEINS; MEMBRANE TRANSPORT; MEMBRANES; METABOLITES; MICROORGANISMS; Molecular and cellular biology; Monosaccharide Transport Proteins - metabolism; MONOSACCHARIDES; NUCLEONS; ORGANIC COMPOUNDS; PEPTIDE HYDROLASES; PROTEIN STRUCTURE; PROTEINS; PROTONS; RADIATION EFFECTS; RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT; REACTION KINETICS; RECEPTORS; SACCHARIDES; SERINE PROTEINASES; TRACER TECHNIQUES; TRITIUM COMPOUNDS; TRYPSIN; Trypsin - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)92958-3

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.