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Proteolytic susceptibility of both isolated and bound light chains from various myosins to myopathic hamster protease
Abstract:
Myopathic hamster protease was incubated with turkey gizzard, scallop adductor, and Loligo mantle retractor myosins in order to establish if the regulatory light chain could be selectively digested. In contrast to cardiac or skeletal muscle myosin in which almost all of the regulatory light chain is degraded, these light chains from smooth and invertebrate muscle myosins were remarkably resistant to proteolysis. In the case of scallop myosin, increasing the protease to myosin ratio resulted in comparable digestions of both the regulatory and essential light chains regardless of the presence of Mg2+. The isolated light chains on the other hand were readily digested into smaller fragments. In addition, it was observed that the myosin heavy chains were extremely sensitive and that it was possible to cleave them quantitatively to produce a new band moving with a mobility on SDS gels corresponding to an Mr of approximately 150,000. This was again at variance with cardiac or skeletal myosin where the breakdown of the heavy chains was shown to be minimal. In spite of the significant extent of heavy chain cleavage, gizzard myosin appears to maintain its tertiary structure as demonstrated by sedimentation velocity and equilibrium ultracentrifugation analysis. Moreover, upon examination by electron microscopy, both intact and cleaved gizzard myosin revealed the characteristic folded structure which had a sedimentation rate of about 10 S when dialyzed into a low salt, Mg X ATP-containing buffer. The effects and implications of such modifications on catalytic activities of gizzard, scallop, and Loligo myosins are discussed in detail.
Insights
Smooth and invertebrate muscle myosins show resistance to protease digestion of regulatory light chains, unlike cardiac and skeletal muscle myosins. Myosin heavy chains are sensitive to cleavage, yet gizzard myosin retains its structure.
Area of Science:
- Biochemistry
- Molecular Biology
- Muscle Physiology
Background:
- Myosin, a key motor protein, comprises heavy and light chains, with regulatory light chains (RLCs) controlling muscle contraction.
- Proteolytic digestion studies are crucial for understanding myosin structure-function relationships.
- Differences in myosin composition across muscle types suggest varied functional properties.
Purpose of the Study:
- To investigate the susceptibility of regulatory light chains from smooth (gizzard) and invertebrate (scallop, Loligo) muscle myosins to proteolytic digestion.
- To compare the proteolytic digestion patterns of these myosins with those of cardiac and skeletal muscle myosins.
- To analyze the structural integrity and functional implications of myosin heavy chain cleavage.
Main Methods:
- Incubation of turkey gizzard, scallop adductor, and Loligo mantle retractor myosins with myopathic hamster protease.
- Analysis of protein digestion patterns using SDS-PAGE.
- Structural integrity assessment via sedimentation velocity and equilibrium ultracentrifugation.
- Electron microscopy of intact and cleaved gizzard myosin.
Main Results:
- Regulatory light chains from smooth and invertebrate myosins were highly resistant to proteolysis, contrasting with cardiac/skeletal myosins.
- Scallop myosin's regulatory and essential light chains were digested comparably at higher protease concentrations.
- Myosin heavy chains were highly sensitive to cleavage, yielding a ~150,000 Mr fragment, unlike the minimal breakdown in cardiac/skeletal myosins.
- Despite extensive heavy chain cleavage, gizzard myosin maintained its tertiary structure and characteristic folded conformation.
Conclusions:
- Smooth and invertebrate muscle myosins possess distinct light chain structures conferring resistance to specific proteases.
- Myosin heavy chain cleavage can occur extensively without compromising overall myosin structure, particularly in gizzard myosin.
- These findings highlight significant structural and functional differences in myosin across various muscle types, impacting their catalytic activities.