Isolation, Characterization and Phosphorylation Pattern of the Troponin Complexes TI2C and I2C
| Autor: | Knut Feldmann, Jakob E. Sperling, Ulrike Jahnke, Ludwig M. G. Heilmeyer, Helmut E. Meyer |
|---|---|
| Rok vydání: | 1979 |
| Předmět: |
Magnetic Resonance Spectroscopy
Protein Conformation Stereochemistry Protein subunit Muscle Proteins macromolecular substances Biochemistry Troponin C Troponin I Animals Amino Acids Phosphorylation Gel electrophoresis Chromatography biology Troponin T Viscosity Chemistry Muscles musculoskeletal system Troponin Molecular Weight Sedimentation coefficient Kinetics Sedimentation equilibrium biology.protein Calcium Rabbits Peptide Hydrolases Protein Binding |
| Zdroj: | European Journal of Biochemistry. 101:581-592 |
| ISSN: | 1432-1033 0014-2956 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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