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The myosin filament. XII. Effect of MgATP on assembly
Abstract:
The effect of MgATP on myosin filament assembly has been studied. Filaments were assembled by a standard dilution procedure involving two steps, dilution from 0.6 to 0.3 M KCl and from 0.3 to 0.15 M KCl with a different rate of dilution in each step. This standard dilution procedure gives filaments which are structurally similar to native filaments in that they have a sharp length distribution around 1.5 micron, a diameter of 16 nm and they vary in length with KCl concentration in a similar manner to native filaments. The addition of 1 mM MgATP leads to a sharpening of the length distribution around 1.5 micron without change in the 16 nm diameter. Filaments assembled by dialysis or by rapid dilution are not similarly affected by the presence of MgATP indicating that the standard dilution procedure produces filaments which are more closely similar to native filaments than those produced by these other methods. MgAMPPNP and magnesium pyrophosphate have the same effect as MgATP thus eliminating the possibility that phosphorylation of the myosin is involved in the effect. The effect of MgATP is not directly related to its binding to the active site of the myosin molecule since a 500:1 mole ratio of MgATP to myosin is required for the effect. It is therefore likely that the effect of MgATP is related to other binding sites on the myosin molecule. The presence of MgATP leads to molecular rearrangements which finely tune the molecular organization of the filaments formed by the standard dilution procedure in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Magnesium ATP (MgATP) refines myosin filament assembly, sharpening length distribution without altering diameter. This effect, observed with MgATP analogs, suggests binding to non-active sites influences filament organization.
Area of Science:
- Biochemistry
- Molecular Biology
- Muscle Physiology
Background:
- Myosin filament assembly is crucial for muscle contraction.
- Understanding factors influencing filament structure is key to muscle function.
- Previous methods produced filaments with less defined structures.
Purpose of the Study:
- To investigate the effect of MgATP on myosin filament assembly.
- To compare MgATP's influence on filaments assembled by different methods.
- To elucidate the mechanism by which MgATP affects filament structure.
Main Methods:
- Myosin filaments were assembled using a two-step dilution procedure.
- Filament length distribution and diameter were analyzed.
- Comparison of assembly methods (dilution, dialysis, rapid dilution) in the presence of MgATP.
- Testing of MgATP analogs (MgAMPPNP, magnesium pyrophosphate).
Main Results:
- A standard dilution procedure yielded native-like myosin filaments (1.5 micron length, 16 nm diameter).
- 1 mM MgATP sharpened length distribution around 1.5 micron without changing diameter.
- MgATP's effect was not observed with dialysis or rapid dilution assembly methods.
- MgATP analogs produced similar effects, ruling out phosphorylation.
- High MgATP:myosin ratio suggests binding to non-active sites.
Conclusions:
- MgATP significantly refines myosin filament structure during assembly via standard dilution.
- The standard dilution procedure yields filaments more structurally similar to native filaments.
- MgATP's effect is likely mediated by binding to non-active sites on myosin, influencing molecular organization.