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

A polyclonal antibody was generated against a peptide corresponding to a re gion opposite the regulatory face of glycogen phosphorylase b (P-b), provid ing a probe for detecting and quantifying P-b when it is bound to its activ ating kinase, phosphorylase kinase (PhK). Using both direct and competition enzyme-linked immunosorbent assays (ELISAs), we have measured the extent o f direct binding to PhK of various forms of phosphorylase, including differ ent conformers induced by allosteric effecters as well as forms differing a t the N-terminal site phosphorylated by PhK. Strong interactions with PhK w ere observed for both P-b', a truncated form lacking the site for phosphory lation, 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 Mg2+, or by Ca2+ (both being activators of PhK). Our results suggest that the presence and conform ation 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 al so interact with PhK. Direct ELISAs detected the binding of P-b by a trunca ted form of the catalytic gamma subunit of PhK, consistent with the necessa ry interaction of PhK's catalytic subunit with its substrate P-b. In contra st, 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 holo enzyme may involve more than just its catalytic core. The sum of our result s suggests that structural Features outside the catalytic domain of PhK and outside the phosphorylatable region of P-b may both be necessary for the m aximal interaction of these two proteins.