1995 Fiscal Year Final Research Report Summary
The mutation of smooth muscle myosin and the sliding mechanism of actin filaments
| Project/Area Number |
06680663
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Biophysics
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| Research Institution | National Cardiovascular Center Research Institute |
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Principal Investigator |
ONISHI Hirofumi National Cardiovascular Center Research Institute, Department of Structural Analysis, Section Chief, 循環器形態部, 室長 (80092542)
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| Project Period (FY) |
1994 – 1995
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| Keywords | Smooth muscle myosin / Protein expression / Sliding movement of actin filaments / Baculovirus |
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| Research Abstract |
In the muscle contraction, myosin functions as the molecular motor that provides the sliding movement of actin filaments. The final goal of this research project is to know which amino acid residues within myosin heads are essential for the sliding of actin filaments. Site-directed mutagenesis using gene engineering is nowadays the most popular technology to investigate the functional role of amino acid residues. In the first year of the project, we developed an expression system of smooth muscle heavy meromyosin. A part of the smooth muscle myosin gene was inserted into baculovirus by gene engineering and cultured insect cells were infected with the engineered virus to express heavy meromyosin. Heavy meromyosin was extracted from the infected insect cells by hypotonic media, co-precipitated with actin filaments, and then purified by ionic exchange chromatography. The purified heavy meromyosin was essentially same in its folding and its enzymatic activity as the heavy meromyosin prepared from smooth muscle myosin after the proteolytic digestion. In the second year, I introduced several mutations to the heavy chain gene of smooth muscle heavy meromyosin. I purified and characterized nine different mutant heavy meromyosins. Some of them showed interesting properties :
1.The mutated (Trp546Ser and Phe547His) heavy meromyosin was minimally activated for its ATPase activity by actin and did not decorate actin well. I thus suggest that Trp^<546> and Phe^<547> are important participants in the hydrophobic actin-myosin interaction.
2.The ATPase of the mutated (Lys845Glu and Lys847Glu) heavy meromyosin was activated by actin and sensitive to the light-chain phosphorylation. Although Lys^<845> of the heavy chain is known to be a binding site of the regulatory light chain, I suggest that Lys^<845> and Lys^<847> are not essential for the phosphorylation-mediated regulation.
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