31P nuclear magnetic resonance studies of glycogen phosphorylase from rabbit skeletal muscle: ionization states of pyridoxal 5'-phosphate.

31P nuclear magnetic resonance (NMR) at 72.8 MHZ has been used to study glycogen phosphorylase from rabbit muscle (1,4-alpha-D-glucan:orthophosphate alpha-glucosyltransferase, EC 2.4.1.1) at concentrations as low as 25 mg/ml, using a WH-180 wide-bore superconducting spectrometer. The use of a thio analogue for 5'-AMP and arsenate for inorganic phosphate allowed the observation of three distinct forms of enzyme-bound pyridoxal 5'-phosphate at --0.2 ppm (Form I), --2 to --3 ppm (Form II), and --3.5 ppm (Form III) relative to triethylphosphate. Conversion of I to III occurs by activation of phosphorylase either by formation of a ternary complex of phosphorylase b with effector and arsenate or, more efficiently, by direct phosphorylation to give the a form of the enzyme. The ionization state and exposure to solvent of each of the three forms is inferred from the 31P NMR data.