Effect of ionic strength on the sedimentation of glycogen phosphorylase a.

Glycogen phosphorylase has been isolated from rabbit muscle in two different molecular forms, phosphorylase a, a tetramer with a molecular weight of 495,000 (2-4), and phosphorylase b, a dimer with a molecular weight of 242,000 (3-5). The dimerization of the protein that occurs in the conversion of phosphorylase 6 to a with phosphorylase kinase, Mg++, and ATP has been found to result from the incorporation of four phosphoryl groups per mole of phosphorylase a (6). Although dimerization appears to follow phosphorylation closely (6), the exact relationship between phosphorylation and dimerization is not clear. The possibility that dimerization occurs through formation of a phosphodiester bridge appears remote since tryptic attack of phosphorylase a liberates all the protein-bound phosphate as a monoester with serine (7). Furthermore, modification of cysteinyl residues of phosphorylaee a by p-mercuribenzoate results in the cleavage of the molecule into four subunits (4). I f the phosphoryl groups are directly involved in the dimerization process, this also cannot be explained by chelation of four phosphoryl groups by a metal introduced in the phosphorylase b to a reaction (6). Tryptic attack of phosphorylase a shows that the portion of the peptide chain at the site of phosphorylation is highly positively cha,rged, and it has been suggested that dimerization follows phosphorylation by neutralization of the charge of this positive site, which would allow an interaction between interpeptide chains that were previously electrostatically repulsed (7). The present work was undertaken to determine whether the phosphoryl groups or some other ionic groups of phosphorylase a were important for the stabilization of the tetrameric form of this enzyme. The results report.ed here show that phosphorylase a is sensitive to ionic strength and at high salt concentrations dissociates into a dimeric form of the enzyme that is not identical with phosphorylase b. The degree of structural change produced by solutions of high ionic strength is dependent upon pH, protein concentration, and the type of salt employed.