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J. Biochem, 1994, Vol. 115, No. 2 351-355
© 1994 Japanese Biochemical Society

research-article

Purification and Characterization of the 1, 200-kDa Subfragment of Connectin Filaments Produced by 0.1 mM Calcium Ions1

Ryoichi Tanabe2, Ryuichi Tatsumi and Koui Takahashi3

Meat Science Laboratory, Department of Animal Science, Faculty of Agriculture, Hokkaido University Kita-ku, Sapporo, Hokkaido 060

3 To whom correspondence should be addressed.

When myofibrils prepared from chicken leg muscle were treated with a solution containing 0.1 mM CaCl2 and 30 µg/ml of leupeptin, {alpha}-connectin, which exists as a longitudinal thin filament in a sarcomere, was split into ß-connectin and {alpha} 1, 200-kDa subfragment. The native subfragment was successfully purified without using any denaturant: It was extracted with 1 M KI solution from the Ca-treated myofibrils and purified by TSKge1 G6000PW column chromatography. About 10 mg of the subfragment was yielded from 100 g of starting muscle. Using immunofluorescence microscopy and immunoelectron microscopy, we show here that polyclonal antibodies against the 1, 200-kDa subfragment bind to the Z-disk and the epitope, which is about 0.34 um apart from the Z-disk at a sarcomere length of 2.6 µm; the 1, 200-kDa subfragment constitutes the proximal region of connectin filaments. Purified {alpha}-actinin decorated {alpha}-connectin and the 1, 200-kDa subfragment on nitrocellulose blots of myofibrillar proteins separated by SDS-PAGE. Therefore, we conclude that connectin filaments are anchored to the Z-disk by the binding of the 1, 200-kDa subfragment to {alpha}-actinin.

1This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.

2Present address: Department of Animal Products, National Institute of Animal Industry, 2 Ikenodai, Kukizaki-cho, Inashiki-gun, Ibaraki 305.


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