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.
Kimura
S
S
Department of Biology, Faculty of Science, Chiba University, Japan.
Matsuura
T
T
Ohtsuka
S
S
Nakauchi
Y
Y
Matsuno
A
A
Maruyama
K
K
D000199:Actins / Q000032:analysis / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000906:Antibodies; D064211:Connectin; D004549:Elastin / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D016253:Microscopy, Immunoelectron; D009124:Muscle Proteins / Q000032:analysis* / Q000378:metabolism; D009132:Muscles / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D009218:Myosins / Q000032:analysis / Q000378:metabolism; D011494:Protein Kinases; D011817:Rabbits
D000199:Actins; D000596:Amino Acids; D000906:Antibodies; D064211:Connectin; D009124:Muscle Proteins; D004549:Elastin; D011494:Protein Kinases; D009218:Myosins