title: A new in vitro motility assay technique to evaluate calcium sensitivity of the cardiac contractile proteins.
abstract: We attempted to introduce calcium regulation into in vitro motility assay. Cardiac thin filament was reconstituted from actin and tropomyosin-troponin complex purified from rat myocardium separately. Double staining of the filaments showed tropomyosin-troponin complex was integrated along actin filaments homogeneously. The reconstituted thin filaments were made to slide on cardiac myosin fixed on a glass coverslip in the presence of MgATP while varying free Ca2+ concentration of the medium ([Ca2+]). Filaments showed only Brownian motion when [Ca2+] was below 10(-6.4) M. However, filaments slid at a constant velocity when [Ca2+] exceeded 10(-6.4) M, showing that the sliding was regulated in an on-off manner. The threshold [Ca2+] increased to 10(-5.0) M under acidic conditions, indicating a decrease in Ca2+ sensitivity of the contractile proteins. Simple actin filaments slid at a constant velocity independently of [Ca2+], demonstrating that the regulatory proteins were responsible for this on-off manner regulation. This new assay technique may be a powerful tool to directly evaluate the Ca2+ sensitivity of the contractile apparatus and to investigate how cardiac contraction is regulated by Ca2+.
authors.affiliation: Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Sata
authors.forename: H
authors.initials: H
authors.lastname: Yamashita
authors.forename: S
authors.initials: S
authors.lastname: Sugiura
authors.forename: H
authors.initials: H
authors.lastname: Fujita
authors.forename: S
authors.initials: S
authors.lastname: Momomura
authors.forename: T
authors.initials: T
authors.lastname: Serizawa
mesh_terms: D000818:Animals; D002118:Calcium / Q000502:physiology*; D003285:Contractile Proteins / Q000502:physiology*; D005456:Fluorescent Dyes; D006321:Heart / Q000502:physiology; D008856:Microscopy, Fluorescence; D009200:Myocardial Contraction / Q000502:physiology*; D009206:Myocardium / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar
chemical_list: D003285:Contractile Proteins; D005456:Fluorescent Dyes; D002118:Calcium