Interaction of Phosphofructokinase with the Fluorescent Probe 2-(<em>N</em>-Methylanilino)naphthalene-6-sulphonate.

The interaction of phosphofructokinase from rabbit skeletal muscle with 2-(N-methylanilino)-naphthalene-6-sulphonate was studied. The fluorescence maximum of the probe was shifted to 430 nm and was enhanced by the enzyme. The intensity of emitted light was reduced by the presence of either one of the substrates, fructose 6-phosphate or ATP. The reduction of fluorescence by fructose 6-phosphate was accompanied by a decrease in the number of binding sites for the probe on the protein. The number of binding sites for the probe was also dependent on the concentration of the enzyme. Decrease of enzyme concentration resulted in relatively higher fluorescence and in rise of the number of binding sites. Phosphofructokinase is inactivated after dilution as well in the absence as in the presence of the probe as a consequence of dissociation into inactive subunits. In the presence of the probe, the loss in activity followed the same kinetics as the increase in fluorescence. The rise in the number of probe binding sites, therefore, is most probably due to the dissociation of the enzyme. The effect of substrates on fluorescence intensity was used to follow inter. actions of the enzyme with its substrates. At low substrate concentrations the fluorescence changes were dependent on the concentration of the enzyme. At higher enzyme levels, higher ATP but lower fructose 6-phosphate concentrations were required to obtain half-maximal effects, thus suggesting that the binding of the substrates is dependent on the association state of the enzyme. [ABSTRACT FROM AUTHOR]