PURIFICATION AND CHARACTERIZATION OF THE 1,200-KDA SUBFRAGMENT OF CONNECTIN FILAMENTS PRODUCED BY 0.1 MM CALCIUM-IONS

When myofibrils prepared from chicken leg muscle were treated with a s olution containing 0.1 mM CaCl2 and 30 mu g/ml of leupeptin, alpha-con nectin, which exists as a longitudinal thin filament in a sarcomere, w as split into beta-connectin and a 1,200-kDa subfragment. The native s ubfragment was successfully purified without using any denaturant: It was extracted with 1M KI solution from the Ca-treated myofibrils and p urified by TSKgel G6000PW column chromatography. About 10 mg of the su bfragment was yielded from 100 g of starting muscle. Using immunofluor escence microscopy and immunoelectron microscopy, we show here that po lyclonal antibodies against the 1,200-kDa subfragment bind to the Z-di sk and the epitope, which is about 0.34 mu m apart from the Z-disk at a sarcomere length of 2.6 mu m; the 1,200-kDa subfragment constitutes the proximal region of connectin filaments. Purified alpha-actinin dec orated alpha-connectin and the 1,200-kDa subfragment on nitrocellulose blots of myofibrillar proteins separated by SDS-PAGE. Therefore, we c onclude that connectin filaments are anchored to the Z-disk by the bin ding of the 1,200-kDa subfragment to alpha-actinin.