Phosphorylase Kinase from Rabbit Skeletal Muscle Identification of the Calmodulin‐Binding Subunits

Abstract

Phosphorylase kinase has the structure (αβγδ)₄ where the δ‐subunit is identical to the calciumbinding protein termed calmodulin [Shenolikar et al. 1979) Eur. J. Biochem. 100, 329–337]. The δ‐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 δ‐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 δ‐subunit was associated with the γ‐subunit. This finding was confirmed by cross‐linking experiments with dimethylsuberimidate which resulted in the formation of a γδ complex. Phosphorylase kinase was shown to bind one additional molecule of calmodulin per αβγδ unit, termed the δ‐subunit. Glycerol gradient centrifugation in the presence of [¹⁴C]calmodulin indicated that the interaction of the δ‐subunit with phosphorylase kinase only occurred in the presence of calcium ions, and that the K_d value was near 0.01 μM. This was similar to the concentration of δ‐subunit which produced half‐maximal activation. The δ‐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 δ‐subunit and [¹⁴C]calmodulin, and the calcium‐dependcnt binding of the δ‐subunit allowed cross‐linking experiments to be used which demonstrated that the δ‐subunit was bound to both the α and β subunits. This result was supported by the finding that selective proteolysis of either the δ‐subunit, or the α and β subunits, decreased or abolished the ability of phosphorylasc kinase to bind to calmodulin‐Sepharose. The roles of the different subunits in the regulation of phosphorylase kinase activity are discussed.