The interaction of ligands with chemically modified phosphorylase b.
Phosphorylase b which had been inactivated with 5-diazo1H-tetrazole was specifically labelled with 4-iodoacetamidosalicylic acid (a fluorescent probe) or with N-(1-oxyl-2,2,6,6,-tetramethyl-4-piperidinyl)iodoacetamide (a spin label probe) so that the binding of ligands and accompanying conformational changes could be determined by fluorescence or electron spin resonance changes, respectively. The allosteric effector, AMP, causes conformational changes similar to those caused in the native enzyme. The affinity of binding of phosphate or AMP to the inhibited protein is the same as for the unmodified protein. The heterotropic interactions between glucose-1-phosphate or glycogen and AMP are much less in the inactivated enzyme than in unmodified phosphorylase. Using a light scattering assay, it is shown that the modified enzyme binds to glycogen less strongly than the native protein. Phosphorylase b which had been inactivated by carbodimide in the presence of glycine ethyl ester, resulting in the modification of one or more carboxyl groups, was labelled with the spin label probe described above. The modified enzyme has an affinity for AMP similar to that of the native enzyme. AMP binding to the modified enzyme is tightened by glycogen, weakened by glucose-6-phosphate and is unaffected by glucose-1-phosphate. The actions of 5-diazo-1H-tetrazole and carbodimide on phosphorylase are discussed in the light  of the above observation.
D000249:Adenosine Monophosphate; D000818:Animals; D001393:Azoles; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D006003:Glycogen; D007460:Iodoacetamide; D007461:Iodoacetates; D008024:Ligands; D008027:Light; D009132:Muscles / Q000201:enzymology; D006005:Phosphorylases / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D012459:Salicylates; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013113:Spin Labels; D013777:Tetrazoles
D001393:Azoles; D007461:Iodoacetates; D008024:Ligands; D012459:Salicylates; D013113:Spin Labels; D013777:Tetrazoles; D000249:Adenosine Monophosphate; D006003:Glycogen; D006005:Phosphorylases; D007460:Iodoacetamide