ISOLATION, CHARACTERIZATION AND PHOSPHORYLATION PATTERN OF THE TROPONIN COMPLEXES TI2C AND I2C

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. Copyright © 1979, Wiley Blackwell. All rights reserved