Characterization and localization of α-connectin (titin 1): An elastic protein isolated from rabbit skeletal muscle

Abstract

A simplified procedure to isolateα-connectin (titin 1, TI), a gigantic elastic protein, from rabbit skeletal muscle is described. A rapid column chromatography step to concentrateα-connectin is introduced. Separation ofα-connectin fromβ-connectin is introduced. Separation ofα-connectin fromβ-connectin (titin 2, TII) in the presence of 4 M urea at pH 7.0 did not cause any change in the secondary structure ofα-connectin as judged by circular dichroic spectra. Ultraviolet absorption spectra and the amino acid composition ofα-connectin (MW, approximately 3×10⁶) were similar to those of its proteolytic product,β-connectin (MW, approximately 2×10⁶). Circular dichroic spectra suggested that bothα- andβ-connectin consist of 60%β-sheet and 30%β-turn. It thus appears that the whole elastic filament of connectin has a foldedβ-strand structure. Proteolysis ofα-connectin by calpain resulted in formation ofβ-connectin and smaller peptides. Theα-connectin interacted with both myosin and actin filaments similarly toβ-connectin. Polyclonal antibodies raised against 1200 kDa peptides obtained from aged rabbit skeletal myofibrils reacted withα-connectin (titin 1, TI) but only weakly withβ-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 SMI whose position depends on sarcomere length. It thus appears thatβ-connectin extends from the edge of M-line to the above epitope region in the I-band.