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Effects of phosphorylated and unphosphorylated C-protein on cardiac actomyosin ATPase
Insights
Cardiac C-protein, a thick filament component, regulates muscle contraction. Unphosphorylated C-protein significantly enhances actin-activated myosin ATPase activity, suggesting a role in cardiac muscle function.
Area of Science:
- Muscle Physiology
- Biochemistry
- Cardiac Muscle Biology
Background:
- C-protein is a key component of striated muscle thick filaments.
- C-protein undergoes reversible phosphorylation in the heart.
- C-protein phosphorylation levels correlate with cardiac relaxation rates, suggesting a regulatory role.
Purpose of the Study:
- To investigate the functional role of C-protein in cardiac muscle contraction.
- To determine the effects of phosphorylated and unphosphorylated C-protein on myosin ATPase activity.
Main Methods:
- Preparation of DEAE-Sephadex-purified myosin.
- Assay of actin-activated myosin ATPase activity with varying concentrations of C-protein.
- Measurement of light-scattering and actomyosin precipitation.
- Analysis of kinetic parameters (Vmax and Km).
Main Results:
- Unphosphorylated C-protein dose-dependently stimulated actin-activated myosin ATPase activity up to 3.2-fold.
- Phosphorylated C-protein showed a lesser stimulation (2.5-fold).
- C-protein increased Vmax and decreased Km, indicating enhanced actin-myosin interaction.
- C-protein promoted actomyosin aggregation and precipitation, with unphosphorylated C-protein having a greater effect.
Conclusions:
- C-protein significantly stimulates actin-activated myosin ATPase activity.
- The stimulation is primarily mediated by enhancing stable actin-myosin filament aggregation.
- These findings support a role for C-protein in regulating cardiac muscle contraction dynamics.
Abstract:
C-protein, a component of the thick filaments of striated muscles, is reversibly phosphorylated and dephosphorylated in heart. It has been hypothesized that C-protein may be involved in regulating contraction, because the extent of C-protein phosphorylation correlates with the rate of cardiac relaxation. To test this hypothesis, the effects of phosphorylated and unphosphorylated C-protein on the actin-activated ATPase activity of myosin filaments prepared from DEAE-Sephadex-purified myosin were examined. Unphosphorylated C-protein (0.1 microM to 1.5 microM) stimulated actin-activated myosin ATPase activity in a dose-dependent manner. With a myosin: C-protein molar ratio of approximately 1, actin-activated myosin ATPase activity was elevated up to 3.2 times that of the control. Phosphorylated C-protein (2.5 mol PO4/mol C-protein) stimulated the activity somewhat less (2.5 times that of control). The stimulation of ATPase activity by C-protein was due to an increase in the Vmax value (from 0.25/second to 0.62/second) and a decrease in the Km value (from 11.9 microM to 6.7 microM). The addition of C-protein to actomyosin solutions produced an increase in the light-scattering of the actomyosin solution and a distinct precipitation of the actomyosin with time. Phosphorylated C-protein had a smaller effect on light-scattering than dephosphorylated C-protein. C-protein had a negligible effect on Ca-ATPase, EDTA-K-ATPase, or Mg-ATPase activities in the absence of actin. C-protein had only small effects on the actin-activated ATPase of heavy meromyosin. These results suggest that C-protein stimulates actin-activated myosin ATPase activity by enhancing the formation of stable aggregates between actin and myosin filaments.