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Conformation, filament assembly, and activity of single-headed smooth muscle myosin
1Division of Chemistry, Graduate School of Science, Hokkaido University, Kita-ku, Sapporo, 060-0810, Japan.
Abstract:
Single-headed myosin was prepared by digestion of porcine aorta smooth muscle myosin with Staphylococcus aureus V8 protease in the presence of actin. The single-headed myosin preparation contained intact light chains, a rod fragment of a heavy chain, and a heavy chain of which only a minor fraction contained a nick in the head segment. Below 0.2 M NaCl, the single-headed myosin showed a decrease in Ca2+-ATPase activity and an increase in the elution time on gel filtration HPLC in a phosphorylation-dependent manner, indicating a phosphorylation-dependent conformational transition between the extended and folded forms. These conformations were confirmed by electron microscopic observation of rotary-shadowed samples of single-headed myosin. However, the conformational transition of single-headed myosin occurred in a narrower range with lower salt concentrations than that of double-headed myosin. The filament assembly of single-headed myosin was thus facilitated and phosphorylation-independent. The single-headed myosin also showed high actin-activated ATPase activity independent of phosphorylation. These results indicate that the two-headed structure of smooth muscle myosin is not essential for the conformational transition, but is required for the phosphorylation-dependent regulation of enzymatic activity and filament assembly.
Insights
Single-headed myosin undergoes phosphorylation-dependent conformational changes, but its two-headed structure is crucial for regulating enzymatic activity and filament assembly in smooth muscle.
Area of Science:
- Biochemistry
- Molecular Biology
- Muscle Physiology
Background:
- Smooth muscle myosin, a key motor protein, plays a vital role in muscle contraction.
- Phosphorylation of myosin light chains regulates smooth muscle function.
- Understanding the structural requirements for myosin regulation is essential for elucidating muscle physiology.
Purpose of the Study:
- To investigate the role of the two-headed structure of smooth muscle myosin in its conformational transitions and regulatory mechanisms.
- To determine if the two-headed structure is essential for phosphorylation-dependent regulation of ATPase activity and filament assembly.
Main Methods:
- Preparation of single-headed myosin from porcine aorta smooth muscle myosin using Staphylococcus aureus V8 protease digestion.
- Analysis of single-headed myosin conformation using gel filtration HPLC and electron microscopy.
- Assay of Ca2+-ATPase activity and actin-activated ATPase activity.
- Investigation of filament assembly properties.
Main Results:
- Single-headed myosin exhibited a phosphorylation-dependent conformational transition between extended and folded forms, confirmed by electron microscopy.
- This transition occurred at lower salt concentrations and in a narrower range compared to double-headed myosin.
- Filament assembly and actin-activated ATPase activity of single-headed myosin were facilitated and independent of phosphorylation.
- The two-headed structure was found to be non-essential for the conformational transition itself.
Conclusions:
- The two-headed structure of smooth muscle myosin is not essential for the phosphorylation-dependent conformational transition.
- However, the two-headed structure is required for the phosphorylation-dependent regulation of both enzymatic activity and filament assembly.
- These findings highlight the distinct roles of myosin head number and phosphorylation in smooth muscle function.