Characterization and localization of alpha-connectin (titin 1): an elastic protein isolated from rabbit skeletal muscle.

A simplified procedure to isolate alpha-connectin (titin 1, TI), a gigantic elastic protein, from rabbit skeletal muscle is described. A rapid column chromatography step to concentrate alpha-connectin is introduced. Separation of alpha-connectin from beta-connectin is introduced. Separation of alpha-connectin from beta-connectin (titin 2, TII) in the presence of 4 M urea at pH 7.0 did not cause any change in the secondary structure of alpha-connectin as judged by circular dichroic spectra. Ultraviolet absorption spectra and the amino acid composition of alpha-connectin (MW, approximately 3 x 10(6)) were similar to those of its proteolytic product, beta-connectin (MW, approximately 2 x 10(6)). Circular dichroic spectra suggested that both alpha- and beta-connectin consist of 60% beta-sheet and 30% beta-turn. It thus appears that the whole elastic filament of connectin has a folded beta-strand structure. Proteolysis of alpha-connectin by calpain resulted in formation of beta-connectin and smaller peptides. The alpha-connectin interacted with both myosin and actin filaments similarly to beta-connectin. Polyclonal antibodies raised against 1200 kDa peptides obtained from aged rabbit skeletal myofibrils reacted with alpha-connectin (titin 1, TI) but only weakly with beta-connectin (titin 2, TII) in rabbit skeletal muscle. Immunoelectron microscopy and indirect immunofluorescence microscopy revealed that the antibodies bound at the Z-line and at the epitope regions in the I-band near the binding site of a monoclonal antibody SM1 whose position depends on sarcomere length. It thus appears that beta-connectin extends from the edge of M-line to the above epitope region in the I-band.