Studies on Ligand Binding during the Conversion of Phosphorylase b to Phosphorylase a

1 Phosphorylase b may be specifically labelled on one fast reacting sulphydryl group per subunit with 4‐iodoacetamido‐salicylic acid without loss of enzymic activity. 2 The fluorescence intensity of the covalently linked acetamido‐salicylate is altered on the binding of substrate and effector ligands. 3 These fluorescence change were used to derive the “apparent” binding constants of the different ligands and to observe the interaction between them. 4 No change is observed in the fluorescence of the acetamido‐salicylate label when phosphorylase b is converted into phosphorylase a . When the conversion is carried out in the presence of either AMP or glucose 6‐phosphate there are fluoresence changes which reflect the differential ligand binding to the two forms of phosphorylase. The fluorescence method in the presence of ligands therefore provides a continuous assay for the b to a conversion. 5 Studies on the rate of interconversion revealed that the ratio of activities of non‐phosphorylated phosphorylase b kinase at pH 6.8 and 8.4 is over 100 compared to a ratio of 3 for phosphorylated kinase. 6 Experiments carried out in the presence of glucose 6‐phosphate directly demonstrated the presence of an intermediate active form of phosphorylase which binds glucose 6‐phosphate tightly unlike fully phosphorylated phosphorylase a . 7 The association of phosphorylase a dimers to tetramers was detected in a mixture of acetamido‐salicylate‐phosphorylase and 4‐nitrobenzo‐2‐oxa‐1, 3‐diazole phosphorylase. The quenching of the fluorescence of the former by the latter species in the mixed tetramer enabled us to confirm that tetramer formation was slower than the appearance of phosphorylase a activity.