Abstract
When myofibrils prepared from chicken leg muscle were treated with a solution containing 0.1mM CaCl2 and 30 μg/ml of leupeptin, α-connectin, which exists as a longitudinal thin fil Pament in a sarcomere, was split into β-connectin and a 1, 200-kDa subfragment. The native subfragment was successfully purified without using any denaturant: It was extracted with 1M KI solution from the Ca-treated myofibrils and purified by TSKgeI G6000PW column chromatography. About 10mg of the subfragment was yielded from 100g 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 μm 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.
