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The myosin filament: XI. Filament assembly
Abstract:
The critical parameters required for the assembly of myosin filaments with a length distribution comparable to that for native myosin filaments were examined. It was found that: Two steps are required in the dilution of a myosin solution from 0.6M KCl to 0.15M KCl. In Step I the KCl concentration is reduced from 0.6 to 0.3M KCl and in Step II from 0.3 to 0.15M KCl. The rate of change of KCl required for Step I is different than that required for Step II. Increasing the total time of dilution in either Step I or II alone leads to an increase in length and a broadening of the length distribution. In Step I assembly of myosin molecules into nonsedimentable units occurs. These may be the basic units from which the filaments are assembled in Step II. Rapid dilution in Step I alone has no effect on the length distribution obtained at 0.15M KCl, but rapid dilution in Step II alone leads to short filaments (about 0.6 micron). Increasing the time of dilution in Step II alone to 3 hrs or 6 hrs gives a bimodal distribution in lengths with one peak at about 0.8 micron and the other at about 2.2 microns. The length distribution obtained at 0.15M KCl is not critically dependent on information contained in the portion of the filament previously assembled in Step II, but is critically dependent on the rate of change of KCl concentration during the assembly of the rest of the filament.
Insights
Two-step dilution of myosin solutions is crucial for assembling native-like myosin filaments. Controlling the rate of potassium chloride (KCl) concentration change in each step optimizes filament length and distribution.
Area of Science:
- Biochemistry
- Molecular Biology
- Muscle Physiology
Background:
- Myosin filament assembly is fundamental to muscle contraction.
- Understanding native myosin filament length distribution is key to elucidating muscle function.
Purpose of the Study:
- To identify critical parameters for assembling myosin filaments with native-like length distributions.
- To investigate the role of dilution rate and steps in myosin filament formation.
Main Methods:
- Controlled two-step dilution of myosin solutions from 0.6M KCl to 0.15M KCl.
- Analysis of myosin filament length and length distribution under varying dilution rates and times.
Main Results:
- Two distinct dilution steps (0.6-0.3M KCl, then 0.3-0.15M KCl) are required.
- Dilution rate in Step II critically influences filament length and distribution, with slower rates yielding bimodal distributions.
- Step I facilitates assembly into nonsedimentable units, potentially serving as precursors for Step II assembly.
Conclusions:
- Myosin filament assembly is sensitive to the kinetics of potassium chloride (KCl) concentration changes.
- The rate of KCl change during the latter stages of assembly (Step II) is paramount for achieving native-like filament lengths and distributions.
- Two-step dilution protocols with controlled rates are essential for reproducible myosin filament assembly.