Phosphorylase kinase, (αβγδ)4, binds in two subsequent steps 2 mol Ca2+/monomer (αβγδ) to high affinity sites, N1a, and 1–2 mol to low affinity sites, N2. Analogously, the holotroponin TI2C binds 2 mol Ca2+ to high affinity and further 2 mol to low affinity sites. In both protein complexes Ca2+ binds to one specific subunit: in phosphorylase kinase to the δ- and in troponin TI2C to the C-subunit. In both proteins the high affinity sites show Ca2+-Mg2+ competition. Cooperative interactions are observable at the low affinity Mg2+ inducible sites in phosphorylase kinase and at the high affinity sites in troponin. The isolated δ subunit binds 2 mol Ca2+ with low affinity in a positive cooperative manner. Ca2+ binding to the high affinity sites inhibits the phosphorylation of the I subunits of troponin but stimulates that of the α and β subunits of phosphorylase kinase. Phosphorylase kinase exhibits three partial activities: A0, A1 and A2. A0 is Ca2+ independent whereas activation of A1 can be correlated to Ca2+ binding to the sites, N1a, and activation of A2 with Ca2+ binding to the sites, N2. Both Ca2+ binding proteins, δ and troponin C, can activate the kinase activity of calsequestrin which is a membrane component of the sarcoplasmic reticulum.

Isolated rabbit skeletal muscle troponin is contaminated with proteases which in the presence of about 10 μM Ca2+ preferentially degrade the troponin T and in about 0.01 μM Ca2+ degrade mainly the troponin I subunit. These proteases can be removed from troponin by adsorption on casein-Sepharosnin TI2C and troponin I2C. The subunit stoichiometry was determined from the separation pattern obtained by gel electrophoresis in the presence of sodium dodecylsulphate; a staining intensity of each isolated subunit per unit weight of 2.10:1.15:1.00 for troponin T: troponin I: troponin C respectively, was found. The content of cysteine was determined by titration with 5,5′-dithiobis(2-nitrobenzoic acid) or performic acid oxidation to be 7.1 or 7.6 mol/100000 g holotroponin complex, respectively. A content of 7.76 mol cysteine/100000 g was calculated for the complex TI2C on the basis of the primary sequences of the troponin subunits. The ratios Cys/Tyr, Cys/Phe and Cys/His do not differ significantly from those calculated for the troponin TI2C. Sedimentation equilibrium at three rotor speeds yields an average molecular weight of 97250 ± 1150 for the holotroponin complex, troponin TI2C; the sedimentation coefficient, S20, w is 3.5 S. From this value and an intrinsic viscosity of 0.018 ml/mg, a molecular weight of 89200 ± 3600 is calculated. Based on a saturation of troponin with 4 mol Ca2+, a molecular weight of 94700 ± 1400 is calculated. A Ca2+-dependent protein kinase incorporates, in the presence of Ca2+(∼ 10 μM), 0.3–1 mol phosphate into the troponin T subunit which contains endogeneously about 1 mol phos-phoserine. At about 0.01 μM free Ca2+, this kinase incorporates phosphate into the troponin I subunit. Phosphate can be additionally incorporated into the 28000-Mr and 14000-Mr breakdown products which are produced during incubation of troponin contaminated with proteases. The catalytic subunit of the cAMP-dependent protein kinase can incorporate phosphate only into the troponin I subunit at low free Ca2+ concentrations to the extent of 0.1–0.6 mol/90200 g protein. The initial rate of phosphate incorporation is half-maximally inhibited at 0.32 μM Ca2+ if the holotroponin TI2C is the substrate; the troponin I2C is half-maximally inhibited at an about fivefold lower free Ca2+ concentration (0.063 μM). The initial phosphorylation rate as a function of the free Ca2+ concentration shows apparent positive cooperativity; Hill coefficients of 1.41 and 1.46 for the complexes troponin TI2C and troponin I2C were determined, respectively. Ca2+ binding shows positive cooperativity only for the two high-affinity Ca2+ binding sites. The 31P nuclear magnetic resonance signal of the phosphate endogeneously present in the troponin T shows identity with phosphoserine and has free rotation. The chemical shift as function of pH of the troponin T phosphoserine is influenced neither by saturation of troponin C with Ca2- nor by the presence of tropomyosin in a 1:1 molar ratio.

The degree of phosphorylation of a protein should correlate with a meaningful change of a functional property if this process is of physiological importance (Krebs and Beavo 1979). Thus, it would require that the introduced phosphate group perturbs interaction of peptide domaines within a polypeptide or between subunits. The phosphate group itself may be used as a reporter group to monitor such perturbations since it yields a [31P] NMR signal which is sensitive to its environment. Therefore the [31P] NMR spectra of phosphorylated skeletal muscle troponin and myosin will be compared with that of phosphorylase a (see also Sperling et al. 1979; Koppitz et al. 1980). It will be shown that these phosphoproteins can be classified into two types according to the appearance of their [31P] NMR signals.