Purification and characterization of the 1, 200-kDa subfragment of connectin filaments produced by 0.1 mM calcium ions

Ryoichi Tanabe, Ryuichi Tatsumi, Koui Takahashi

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Abstract

When myofibrils prepared from chicken leg muscle were treated with a solution containing 0.1 mM CaCl2 and 30 μg/ml of leupeptin, α-connectin, which exists as a longitudinal thin filament in a sarcomere, was split into β-connectin and α 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 α-actinin decorated α-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 α-actinin.

Original languageEnglish
Pages (from-to)351-355
Number of pages5
JournalJournal of biochemistry
Volume115
Issue number2
DOIs
Publication statusPublished - Jan 1 1994
Externally publishedYes

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All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

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