EQUILIBRIUM AND TRANSIENT KINETIC-STUDIES OF THE BINDING OF CYTOCHALASIN-B TO THE L-ARABINOSE-H-COLI - DETERMINATION OF THE SUGAR BINDING-SPECIFICITY OF THE L-ARABINOSE-H+ SYMPORTER( SYMPORT PROTEIN OF ESCHERICHIA)

The kinetics of the binding of cytochalasin B to the L-arabinose-H+ sy mport protein of Escherichia coli have been investigated, using a stra in 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-arabin ose-H+ symporter upon binding the ligand, by stopped-flow fluorescence spectroscopy. The association and dissociation rate constants thus de termined were confirmed by rapid filtration measurements, using [H-3]c ytochalasin B, yielding values of 4.5-6.5 muM-1 . s-1 and 4-5 s-1, res pectively, consistent with K(d) values obtained by measuring equilibri um binding of [H-3]cytochalasin B by dialysis at 22-degrees-C. Titrati on of the protein fluorescence with cytochalasin B yielded a similar b inding site concentration and K(d) value to those obtained in equilibr ium binding studies. All the measurements of binding site concentratio n 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 foll owing order of substrate binding 5-thio-D-glucose > D-fucose > L-arabi nose > 6-deoxy-6-fluoro-D-galactose > D-xylose almost-equal-to 6-deoxy -D-glucose > D-galactose > D-glucose > D-ribose. Neither D-arabinose n or L-fucose had any significant inhibitory effect upon cytochalasin B binding.