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 K(d) of 0.7 - 1.0 muM 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 [H-3]cytochalasin B, yielding values of 4.5-6.5 muM-1 . s-1 and 4-5 s-1, respectively, consistent with K(d) values obtained by measuring equilibrium binding of [H-3]cytochalasin B by dialysis at 22-degrees-C. Titration of the protein fluorescence with cytochalasin B yielded a similar binding site concentration and K(d) 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 almost-equal-to 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.