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

Research output: Contribution to journalArticle

25 Citations (Scopus)

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

Fingerprint

Connectin
Purification
Ions
Calcium
Actinin
Sarcomeres
Myofibrils
Muscle
Microscopic examination
Muscles
Column chromatography
Collodion
Immunoelectron Microscopy
Fluorescence Microscopy
Chromatography
Epitopes
Polyacrylamide Gel Electrophoresis
Chickens
Leg
Antibodies

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Cite this

Purification and characterization of the 1, 200-kDa subfragment of connectin filaments produced by 0.1 mM calcium ions. / Tanabe, Ryoichi; Tatsumi, Ryuichi; Takahashi, Koui.

In: Journal of biochemistry, Vol. 115, No. 2, 01.01.1994, p. 351-355.

Research output: Contribution to journalArticle

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