Phosphorylase kinase has the structure (αβγδ)4 where the δ‐subunit is identical to the calciumbinding protein termed calmodulin. In the presence of calcium ions the enzyme can interact with either a second molecule of calmodulin (termed the δ‐subunit) or with skeletal muscle troponin‐C leading to further enhancement of the activity [Shenolikar et al]. (1979) Eur. J. Biochem. 100, 329–337; Cohen et al. (1979) FEBS Lett. 104, 25–30; Picton et al. (1980) Eur. J. Biochem. (1980) III, 553–561]. In this paper the regulation of the different forms of phosphorylase kinase by calcium ions, the δ‐subunit and troponin have been investigated. The dephosphorylated form, termed phosphorylase kinase b, was found to be almost inactive below 1.0 μM calcium ions. The concentration required for half‐maximal activation, co, 5, for calcium ions was 23 μM at pH 6.8 and 16 μM at pH 8.2. At saturating calcium ions, phosphorylase kinase b was activated fivefold by saturating concentrations of δ‐subunit or the troponin complex. The c0.5 for the δ‐subunit and the troponin complex was 0.018 μM and 1.2 μM respectively at pH 6.8 and 0.012 μM and 1.5 μM respectively at pH 8.2. The c0.5 for calcium ions required for the activation of phosphorylase kinase b by the δ‐subunit was 20 μM at pH 6.8 and 11 μM at pH 8.2. These values were very similar to the c0.5 for calcium ions measured in the absence of the c0.5δ‐subunit. Phosphorylase kinase b was almost inactive at 1.0 μM calcium ions even in the presence of the δ‐subunit. The c0.5 for calcium ions required for the activation of phosphorylase kinase b by the troponin complex was 4.0 μM at pH 6,8 and 2.0 μM at pH 8.2. At calcium ion concentrations below 3.0 μM the activity of phosphorylase kinase b was almost completely dependent on troponin. The activation was 20–30‐fold at saturating concentrations of troponin. Artificial thin filaments made by mixing actin, tropomyosin and the troponin complex at physiological concentrations were just as effective as troponin C or the troponin complex in the activation of phosphorylase kinase b. No activation of phosphorylase kinase b by cardiac troponin‐C was observed at concentrations of 2–20 μM. No activation of phosphorylase kinase b by parvalbumin was detected, even at concentrations as high as 200 μM. Four different parvalbumins corresponding to two distinct evolutionary lineages of the protein were employed in these experiments. Phosphorylase kinase a obtained by phosphorylation with cyclic‐AMP‐dependent protein kinase was 15‐fold more active than phosphorylase kinase b at saturating calcium ions (0.08 mM). The c0.5 for calcium ions of the a‐form was 1.6 μM at pH 6.8 and 0.6 μM at pH 8.2, 15–30‐fold lower than phosphorylase kinase b. Consequently, phosphorylase kinase a was 150‐fold more active than the b‐form at 1.0 μM calcium ions. Phosphorylase kinase a displayed < 5% of its maximal potential activity at 0.1 μM calcium ions and had very little activity below 0.03 μM calcium ions. Phosphorylase kinase a could only be activated slightly by the δ‐subunit (1.3‐fold) or by the troponin complex (1.2‐fold). Phosphorylase kinase a obtained by limited proteolysis with trypsin was 20‐fold more active than the b‐form at saturating calcium ions (0.08 mM). The c0.5 for calcium ions of the a‐form was 0.07 μM at μH 6.8 and 0.05 μM at pH 8.2, 300‐fold lower than phosphorylase kinase h. Con‐ sequently, phosphorylase kinase a′ was 300–400‐fold more active than phosphorylase kinase h at 1.O pM calcium ions. The properties of phosphorylase kinase a′ demonstrated that even a 2% degradation of phosphorylase kinase b would drastically effect plots of activity versus calcium ion concentration. Unlike the b‐form and the a‐form, phosphorylase kinase a′ did not have an absolute requirement for calcium ions and its activity in the absence of calcium ions was 25% of that measured at saturating calcium ions. The δ′‐subunit and troponin had almost no effect on the activity of phosphorylase kinase a′. The majority of the phosphorylase kinase, glycogen synthase and glycogen phosphorylase remained associated with the myofibrillar fraction when skeletal muscle from C3H/He‐mg mice was homogenized in the presence of calcium ions. This did not occur when the muscle was homogenized in the presence of EDTA. Identical results were obtained for glycogen phosphorylase and glycogen synthase using skeletal muscle from ICR/IAn mice which lack the phosphorylase kinase protein. The interaction of phosphorylase kinase with troponin C is therefore not the basis for the calcium‐dependent association of the enzymes of glycogen metabolism with the myofibrils. The following conclusions have been drawn from this work. The binding of three of four molecules of calcium ions to the δ′ ‐subunitis necessary before hosphorylase kinase b and a can be activated by this second molecule of calmodulin. The binding of three or four molecules of calcium to each δ′‐subunit appears to be required for the activation of phosphorylase kinase b. The binding of only one or two molecules of calcium ions to each δ′‐subunit appears to be required for the activation of phosphorylase kinase a or a′. Troponin C is the dominant calcium‐dependent regulator of phosphorylase kinase b at calcium ion concentrations in the micromolar range. It is proposed that troponin C rather than the δ′‐subunit is the physiological activator of phosphorylase kinase b, and that this represents the most important mechanism for coupling glycogenolysis and muscle contraction. In contrast, the δ‐subunit is the dominant calcium‐dependent regulator of phosphorylase kinase a, and calmodulin therefore determines the regulation of phosphorylase kinase in its hormonally activatcd state.

COHEN, P. (1980). The Role of Calcium Ions, Calmodulin and Troponin in the Regulation of Phosphorylase Kinase from Rabbit Skeletal Muscle. European Journal of Biochemistry, 111(2), 563–574. Portico.