| Co-Investigator(Kenkyū-buntansha) |
HARTSHORNE David J. Arizona University, Professor, 生化学栄養食品化学部門, 教授
TOKUMITSU Hiroshi Nagoya University, Assistant professor, 医学部, 助手 (20237077)
WATANABE Masato Nagoya University, Assistant professor, 医学部, 助手 (30220924)
KOBAYASHI Ryoji Nagoya University, Associate professor, 医学部, 助教授 (00020917)
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| Research Abstract |
Myosin phosphorylation-dephosphorylation is the primary Ca^2-mediated regulatory process in smooth muscle. However, recent physiological studies showed that the tension in intact smooth muscle fiber is maintained in spite of the dephosphorylation of myosin, and have suggested that other control mechanisms may exist which modulate the contractile state of the muscle. Can contraction be regulated by protein kinase (s) other than myosin light chain kinase (MLCK), and by Ca2^2-binding proteins other than calmodulin? In this international scientific research program, we have attempted to unify and reconstruct the myosin phosphorylation theory on contractile response of smooth muscle and non-muscle cells, and obtained the following results, according to the schedule.
1) We prepared monoclonal antibodies directed against chicken gizzard MLCK. One of the monoclonal antibody, MM-7 inhibited the kinase activity and the superprecipitation of bovine aortic smooth muscle actomyosin. We also demonstr
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ated the existence of it least 4 subspecies of MLCK in chicken tissues and the heterogeneity of tissue- and species-specific isozyme forms.
2) Caldesmon, an actin and calmodulin binding protein, was phosphorylated by PK-C and calmodulin-dependent protein kinase.
3) The calmodulin-dependent caldesmon kinase was an isozyme of the brain-rich calmodulin-dependent protein kinase II (CaM KII).
4) CaM KII phosphorylated purified myosin light chain at same sites, as MLCK did. Our original CaM KII specific inhibitor, KN-62 inhibited the various agonist-induced contraction in rabbit common carotid arterial strips. CaM KII may be involved in smooth muscle contraction.
5) We detected and purified three new Ca^<2+> binding proteins, using our original compounds affinity chromatography. One was calcyclin and the others were novel Ca^<2+>-binding proteins (tentatively designated calgizzarin and calvasculin). The presence of these Ca^<2+>binding proteins in smooth muscle cells show that novel intracellular Ca^<2+> messenger system (s) may exist. Less
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