CHARACTERIZATION OF AGGREGATES STATES OF GLYCOGEN PHOSPHORYLASES BY GEL ELECTROPHORESIS

The disc gel electrophoretic method developed previously for the characterization of size and/or charge differences in proteins has been applied to glycogen phosphorylase. We have concluded that phosphorylases a and b have the same molecular size but different net charges under the conditions of electrophoresis employed. The enzymes are both dimers with molecular weights of 170,000-180,000 g/mole at pH 8.5, 35°, and a protein concentration of 0.5 mg/ml. Phosphorylase dimer can be dissociated to monomers using sodium dodecyl sulfate or 2 m urea at 4°. The binding of various ligands (adenosine monophosphate, glucose 6-phosphate, and dextrins) changes the relative mobility of phosphorylase. Plots of the logarithm of the relative mobility vs. gel concentration in the presence of ligands yields lines parallel to the line obtained with phosphorylase alone. This behavior is interpreted as an alteration in the net charge of the migrating macromolecule. In the case of dextrin binding, however, an alternative explanation is proposed. A mathematical model is derived which indicates that parallel lines can arise with no change in charge if an equilibrium exists between a slowly migrating protein-ligand complex and free protein. The sizecharge relations of chemically modified forms of phosphorylase (NaBH4-reduced phosphorylase b and apophosphorylase b, phosphorylase b′) have also been determined and are compared with the native enzyme. In addition to characterizing the various aggregated forms of glycogen phosphorylase, this study extends the use of the gel electrophoretic method for analyzing the size-charge relations of proteins to situations involving ligand binding. © 1969, American Chemical Society. All rights reserved.