Direct observation of a central bare zone in a native thick filament isolated from the anterior byssus retractor muscle of Mytilus edulis using fluorescent ATP analogue.
To investigate the existence of a central bare zone in native thick filaments isolated from the anterior byssus retractor muscle (ABRM) of blue mussels (Mytilus edulis), the filaments were observed by fluorescence and dark-field microscopy after being incubated in the presence of Ca2+ with the fluorescent ATP analogue, Cy3-EDA-ATP. Filaments appeared under dark-field illumination as thin rods with tapered ends of length 5-30 microm. Fluorescence microscopy revealed that Cy3-EDA-ATP was bound to these filaments, except near their center. Although the boundary between this central non-fluorescent zone and fluorescent regions was not clearly defined, there was a trend for the width of the central non-fluorescent zone to increase with thick filament length (correlation coefficient = 0.45; n = 142). When Cy3-EDA-nucleotides bound to thick filaments were displaced by excess ATP, fluorescent images disappeared with a rate constant of 0. 024 s-1, close to the turnover rate of Cy3-EDA-ATP by myosin on the native thick filaments. These results indicate that each native thick filament isolated from the ABRM has a central bare zone, but its boundary was not sharply resolved.
Oiwa
K
K
Kansai Advanced Research Center, Communications Research Laboratory, 588-2 Iwaoka, Nishi-ku, Kobe 651-2401. oiwa@crl.go.jp.
Yamaga
T
T
Yamada
A
A
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D000818:Animals; D049872:Bivalvia; D005456:Fluorescent Dyes; D008856:Microscopy, Fluorescence; D009132:Muscles / Q000033:anatomy & histology* / Q000737:chemistry; D009218:Myosins / Q000032:analysis
C112962:2'(3')-O-(N-(2-((Cy3)amido)ethyl)carbamoyl)adenosine triphosphate; D005456:Fluorescent Dyes; D000255:Adenosine Triphosphate; D009218:Myosins