Phosphorylase kinase from rabbit skeletal muscle: identification of the calmodulin-binding subunits.

Abstract

Phosphorylase kinase has the structure (alpha beta gamma delta)4 where the delta-subunit is identical to the calcium-binding protein termed calmodulin [Shenolikar et al. (1979) Eur. J. Biochem. 100, 329--337]. The delta-subunit was tightly bound to phosphorylase kinase in the absence of calcium ions, and its rate of exchange with [14C]calmodulin was only 15% per week. The delta-subunit remained associated with phophorylase kinase in the presence of 8 M urea provided that calcium ions were present and this property enabled electrophoretic techniques to be used which demonstrated that the delta-subunit was associated with the gamma-subunit. This finding was confirmed by cross-linking experiments with dimethylsuberimidate which resulted in the formation of a gamma delta complex. Phosphorylase kinase was shown to bind one additional molecule of calmodulin per alpha beta gamma delta unit, termed the delta'-subunit. Glycerol gradient centrifugation in the presence of [14C]calmodulin indicated that the interaction of the delta'-subunit with phosphorylase kinase only occurred in the presence of calcium ions, and that the Kd value was near 0.01 microM. This was similar to the concentration of delta'-subunit which produced half-maximal activation. The delta'-subunit did not remain associated with phosphorylase kinase in the presence of 8 M urea, either in the presence or absence of calcium ions. The very slow exchange between the delta-subunit and [14C]calmodulin, and the calcium-dependent binding of the delta'-subunit allowed cross-linking experiments to be used which demonstrated that the delta'-subunit was bound to both the alpha and beta subunits. This result was supported by the finding that selective proteolysis of either the alpha-subunit, or the alpha and beta subunits, decreased or abolished the ability of phosphorylase kinase to bind to calmodulin-Sepharose. The roles of the different subunits in the regulation of phosphorylase kinase activity are discussed.