Related Experiment Videos
MgATP specifically controls in vitro self-assembly of vertebrate skeletal myosin in the physiological pH range
Abstract:
The appearances in the electron microscope of rat and rabbit skeletal muscle myosin filaments and rod aggregates, formed in the presence of variable amounts of MgATP, were compared at different pH values. It is shown that small amounts of MgATP, similar to those sufficient to trigger the dissociation of the actomyosin complex, were able to modify the geometry of myosin filaments profoundly in the physiological pH range, whereas the conformation of rod aggregates remained unchanged even in the presence of high concentrations of MgATP. Myosin filaments formed in the absence of MgATP displayed the classical spindle-shaped conformation and variable diameters at all pH values, whereas myosin filaments formed in the presence of MgATP in the physiological pH range had constant diameters, similar to those of natural thick filaments. These filaments of constant diameter frayed, rapidly and reversibly, into two types of subfilaments with respective diameters of 4 to 5 nm and 9 to 10 nm, when the pH of the medium was raised above 7.2. Spindle-shaped myosin filaments and rod aggregates remained unchanged by such small changes in pH. It was possible to change the conformation of preformed spindle-shaped filaments by simply adding MgATP to the medium, but this reaction was slow and took several hours to be completed. Relatively high concentrations of MgATP, similar to those in the living cell, increased the solubility of both myosin filaments and rod aggregates in the alkaline pH range (pH greater than or equal to 7.0). Low pH values (less than or equal to 6.5) and excess free Mg2+ (greater than or equal to 6 to 7 mM) abolished both the specific effect of MgATP on myosin filament conformation and its solubilizing effect on both myosin filaments and rod aggregates. The degree of purity of the myosin preparations and the level of phosphorylation of the LC-2 light chains did not influence filament behaviour noticeably and rat and rabbit myosins behaved similarly.
Insights
Magnesium ATP (MgATP) significantly alters myosin filament structure in skeletal muscle, influencing their diameter and stability. These MgATP-induced changes are pH-dependent and crucial for understanding muscle mechanics.
Area of Science:
- Biochemistry
- Muscle Physiology
- Structural Biology
Background:
- Myosin filaments are essential for muscle contraction.
- The role of MgATP in myosin filament structure and stability is not fully understood.
- Actomyosin complex dissociation is triggered by MgATP.
Purpose of the Study:
- To investigate the effect of MgATP on the conformation of skeletal muscle myosin filaments and rod aggregates.
- To compare the structural changes induced by MgATP at different pH values.
- To understand the influence of MgATP concentration on myosin filament solubility.
Main Methods:
- Electron microscopy of rat and rabbit skeletal muscle myosin filaments and rod aggregates.
- Comparison of structures formed in the presence and absence of MgATP.
- Assessment of structural changes at varying pH levels and MgATP concentrations.
Main Results:
- MgATP profoundly modifies myosin filament geometry in the physiological pH range, resulting in constant diameters.
- Myosin filaments formed with MgATP fray into subfilaments at pH > 7.2.
- Rod aggregates' conformation remains unchanged by MgATP, unlike myosin filaments.
- MgATP increases solubility of myosin filaments and rod aggregates at alkaline pH.
- Low pH or excess Mg2+ abolishes MgATP effects.
Conclusions:
- MgATP plays a critical role in regulating myosin filament structure and stability.
- Myosin filament conformation is sensitive to MgATP and pH, impacting muscle function.
- These findings provide insights into the molecular mechanisms of muscle contraction and myosin assembly.