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

Journal Article · Sun May 05 00:00:00 EDT 1991 · Journal of Biological Chemistry; (USA)

OSTI ID:5642583

Cairns, M T; McDonald, T P; Horne, P; Henderson, P J; Baldwin, S A ^[1]

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1. Univ. of London (United Kingdom)

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-{sup 3}H)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-{sup 3}H)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.

• OSTI ID: 5642583
• Journal Information: Journal of Biological Chemistry; (USA), Vol. 266:13; ISSN 0021-9258
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
GLUCOSE
MEMBRANE TRANSPORT
MEMBRANE PROTEINS
PROTEIN STRUCTURE
METABOLITES
BIOLOGICAL FUNCTIONS
BIOCHEMICAL REACTION KINETICS
CARBOHYDRATES
CELL MEMBRANES
CHEMICAL RADIATION EFFECTS
ESCHERICHIA COLI
GALACTOSE
PROTONS
RECEPTORS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
TRYPSIN
ALDEHYDES
BACTERIA
BARYONS
CELL CONSTITUENTS
ELEMENTARY PARTICLES
ENZYMES
FERMIONS
HADRONS
HEXOSES
HYDROGEN COMPOUNDS
HYDROLASES
ISOTOPE APPLICATIONS
KINETICS
MEMBRANES
MICROORGANISMS
MONOSACCHARIDES
NUCLEONS
ORGANIC COMPOUNDS
PEPTIDE HYDROLASES
PROTEINS
RADIATION EFFECTS
REACTION KINETICS
SACCHARIDES
SERINE PROTEINASES
550201* - Biochemistry- Tracer Techniques
560120 - Radiation Effects on Biochemicals
Cells
& Tissue Culture