Structural features contributing to complex formation between glycogen phosphorylase and phosphorylase kinase.

A polyclonal antibody was generated against a peptide corresponding to a region opposite the regulatory face of glycogen phosphorylase b (P-b), providing a probe for detecting and quantifying P-b when it is bound to its activating kinase, phosphorylase kinase (PhK). Using both direct and competition enzyme-linked immunosorbent assays (ELISAs), we have measured the extent of direct binding to PhK of various forms of phosphorylase, including different conformers induced by allosteric effectors as well as forms differing at the N-terminal site phosphorylated by PhK. Strong interactions with PhK were observed for both P-b', a truncated form lacking the site for phosphorylation, and P-a, the phosphorylated form of P-b. Further, the binding of P-b, P-b', and P-a was stimulated a similar amount by Mg(2+), or by Ca(2+) (both being activators of PhK). Our results suggest that the presence and conformation of P-b's N-terminal phosphorylation site do not fully account for the protein's affinity for PhK and that regions distinct from that site may also interact with PhK. Direct ELISAs detected the binding of P-b by a truncated form of the catalytic gamma subunit of PhK, consistent with the necessary interaction of PhK's catalytic subunit with its substrate P-b. In contrast, P-b' bound very poorly to the truncated gamma subunit, suggesting that the N-terminal phosphorylatable region of P-b may be critical in directing P-b to PhK's catalytic subunit and that the binding of P-b' by the PhK holoenzyme may involve more than just its catalytic core. The sum of our results suggests that structural features outside the catalytic domain of PhK and outside the phosphorylatable region of P-b may both be necessary for the maximal interaction of these two proteins.