Note Added in Proof. It has recently been found that troponin C can substitute for the second molecule of calmodulin in the activation of phosphorylase kinase [Cohen, P., Picton, C. & Klee, C. B. (1979) FEBS Lett. 104, 25–30].
The Role of Calmodulin in the Structure and Regulation of Phosphorylase Kinase from Rabbit Skeletal Muscle
Article first published online: 28 JUN 2008
European Journal of Biochemistry
Volume 100, Issue 2, pages 329–337, October 1979
How to Cite
SHENOLIKAR, S., COHEN, P. T. W., COHEN, P., NAIRN, A. C. and PERRY, S. V. (1979), The Role of Calmodulin in the Structure and Regulation of Phosphorylase Kinase from Rabbit Skeletal Muscle. European Journal of Biochemistry, 100: 329–337. doi: 10.1111/j.1432-1033.1979.tb04175.x
- Issue published online: 28 JUN 2008
- Article first published online: 28 JUN 2008
- (Received April 20, 1979)
Phosphorylase kinase from rabbit skeletal muscle has been shown to possess the structure (αβγδ)4, where the δ-subunit is identical to the calcium binding protein, termed calmodulin. The amount of calmodulin was found to be stoichiometric with the α, β and γ subunits in all preparations of phosphorylase kinase, and was not dissociated from the enzyme even in the presence of 8 M urea, provided that calcium ions were present.
The activity of phosphorylase kinase was increased by the addition of calmodulin to the assay, and half-maximal activation was observed at a molar ratio, calmodulin/phosphorylase kinase, of 20:1. At saturating concentrations of calmodulin, all preparations of phosphorylase kinase had a specific activity of 13.5 ± 1.0 U/mg at pH 8.2. In the absence of calmodulin, the specific activity ranged from 2–7 U/mg, so that the stimulation by calmodulin varied from 2–7-fold with different preparations of phosphorylase kinase. The molecular basis for this variability is discussed.
The stimulation of the activity by calmodulin was prevented by the addition of troponin-I or the antipsychotic drug trifluoperazine, whereas these compounds had little effect on the calcium-dependent activity in the absence of calmodulin. These results demonstrated that the activation by calmodulin was caused by the interaction of a second molecule of calmodulin with phosphorylase kinase. The existence of an additional calmodulin binding site was also indicated by the finding that phosphorylase kinase bound to calmodulin-Sepharose in the presence of calcium ions.
Experiments with ICR/IAn mice which lack muscle phosphorylase kinase activity and C3H/He-mg mice with normal activity, demonstrated that when muscle extracts were fractionated with ammonium sulphate, 90–95% of the calmodulin which precipitated at 0–35% ammonium sulphate was bound to phosphorylase kinase. This information was used to show that at least 35% of the calmodulin in low ionic strength EDTA extracts of rabbit skeletal muscle was bound to phosphorylase kinase.
Muscle extracts from ICR/IAn mice and C3H/He-mg mice had identical myosin light-chain kinase activities, showing that there was not a generalized defect in calmodulin-dependent enzymes in ICR/IAn mice. The skeletal muscle extracts of ICR/IAn mice contained 60% of the calmodulin found in C3H/He-mg mice, and addition of calmodulin did not restore phosphorylase kinase activity to muscle extracts prepared from ICR/IAn mice. This indicated that the lack of phosphorylase kinase activity was not caused by an absence of the calmodulin molecule.
The role of calmodulin in the regulation of muscle phosphorylase kinase activity is discussed.