Equilibrium and transient kinetic studies of the binding of cytochalasin B to the L-arabinose-H+ symport protein of Escherichia coli. Determination of the sugar binding specificity of the L-arabinose-H+ symporter.
The kinetics of the binding of cytochalasin B to the L-arabinose-H+ symport protein of Escherichia coli have been investigated, using a strain that over-produces the symport protein in the cytoplasmic membrane. Equilibrium binding studies revealed a single set of binding sites (2.9-8.9 nmol/mg protein) with a Kd of 0.7-1.0 microM at 22 degrees C. It proved possible to follow the transient kinetics of cytochalasin B binding by measuring the changes in the fluorescence of the L-arabinose-H+ symporter upon binding the ligand, by stopped-flow fluorescence spectroscopy. The association and dissociation rate constants thus determined were confirmed by rapid filtration measurements, using [3H]cytochalasin B, yielding values of 4.5-6.5 microM-1.s-1 and 4-5 s-1, respectively, consistent with Kd values obtained by measuring equilibrium binding of [3H]cytochalasin B by dialysis at 22 degrees C. Titration of the protein fluorescence with cytochalasin B yielded a similar binding site concentration and Kd value to those obtained in equilibrium binding studies. All the measurements of binding site concentration are consistent with a stoichiometry of 1 mol cytochalasin B binding sites/mol L-arabinose-H+ symport protein. Inhibition of both the rate and equilibrium binding of cytochalasin B by sugars indicated the following order of substrate binding 5-thio-D-glucose > D-fucose > L-arabinose > 6-deoxy-6-fluoro-D-galactose > D-xylose approximately 6-deoxy-D-glucose > D-galactose > D-glucose > D-ribose. Neither D-arabinose nor L-fucose had any significant inhibitory effect upon cytochalasin B binding.
Walmsley
A R
AR
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, England.
Petro
K R
KR
Henderson
P J
PJ
D001089:Arabinose / Q000378:metabolism* / Q000494:pharmacology; D001426:Bacterial Proteins / Q000378:metabolism*; D050260:Carbohydrate Metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D003571:Cytochalasin B / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D007700:Kinetics
C016242:AraF protein, E coli; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D003571:Cytochalasin B; D001089:Arabinose