Skip to main content
SpringerLink
Log in

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

  • Papers
  • Published:
Journal of Muscle Research & Cell Motility Aims and scope Submit manuscript

Summary

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Benian, G. M., Kiff, J. E., Neckelmann, N., Moerman, D. G. &Waterston, R. H. (1989) Sequence of an unusually large protein implicated in regulation of myosin activity inC. elegans.Nature 342, 45–50.

    PubMed  Google Scholar 

  • Campbell, D. G., Williams, A. F., Bayley, P. M. &Reid, K. B. M. (1979) Structural similarities between Thy-1 antigen from rat brain and immunoglobulin.Nature 282, 341–42.

    PubMed  Google Scholar 

  • Funatsu, T., Higuchi, H. &Ishiwata, S. (1990) Elastic filaments in skeletal muscle revealed by selective removal of thin filaments with plasma gelsolin.J. Cell Biol. 110, 53–62.

    PubMed  Google Scholar 

  • Fürst, D. O., Osborn, M., Nave, R. &Weber, K. (1988) The organization of titin filaments in the half-sarcomere revealed by monoclonal antibodies in immunoelectron microscopy: a map of ten non-repetitive epitopes starting at the Z-line extends close to the M-line.J. Cell Biol. 106, 1563–72.

    PubMed  Google Scholar 

  • Fürst, D. O., Nave, R., Osborn, M. &Weber, K. (1989) Repetitive titin epitopes with a 42 nm spacing coincide in relative position with known A-band striations also identified by major myosin-associated proteins: an immunoelectron microscopical study on myofibrils.J. Cell Sci. 94, 119–25.

    PubMed  Google Scholar 

  • Horowits, R. &Podolsky, R. J. (1987) The positional stability of thick filaments in activated skeletal muscle depends on sarcomere length: evidence for the role of titin filaments.J. Cell Biol. 105, 2217–23.

    PubMed  Google Scholar 

  • Horowits, R., Maruyama, K. &Podolsky, R. J. (1989) Elastic behavior of connectin filaments during thick filament movement in activated skeletal muscle.J. Cell Biol. 109, 2169–76.

    PubMed  Google Scholar 

  • Hu, D. H., Kimura, S. &Maruyama, K. (1989a) Myosin oligomers as the molecular mass standard in the estimation of molecular mass of nebulin (800 kDa) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.Biomed. Res. 10, 165–8.

    Google Scholar 

  • Hu, D. H., Kimura, S., Kawashima, S. &Maruyama, K. (1989) Calcium activated neutral protease quickly convertsα-connectin toβ-connectin.Zool. Sci. 6, 797–800.

    Google Scholar 

  • Itoh, Y., Suzuki, T., Kimura, S., Ohashi, K., Higuchi, H., Sawada, H., Shimizu, T., Shibata, M. &Maruyama, K. (1988) Extensible and less-extensible domains of connectin filaments in stretched vertebrate skeletal muscle sarcomere as detected by immunofluorescence and immunoelectron microscopy using monoclonal antibodies.J. Biochem. 104, 504–8.

    PubMed  Google Scholar 

  • Kimura, S. &Maruyama, K. (1983a) Preparation of native connectin from chicken breast muscle.J. Biochem. 94, 2083–5.

    PubMed  Google Scholar 

  • Kimura, S. &Maruyama, K. (1983) Interaction of native connectin with myosin and actin.Biomed. Res. 4, 607–10.

    Google Scholar 

  • Kimura, S. &Maruyama, K. (1989) Isolation ofα-connectin, an elastic protein, from rabbit skeletal muscle.J. Biochem. 106, 952–4.

    PubMed  Google Scholar 

  • Kimura, S., Yoshidomi, H. &Maruyama, K. (1984) Proteolytic fragments of connectin cause aggregation of myosin filaments but not of actin filaments.J. Biochem. 96, 1947–50.

    PubMed  Google Scholar 

  • Kurzban, G. P. &Wang, K. (1988) Giant polypeptides of skeletal muscle titin: sedimentation equilibrium in guanidine hydrochloride.Biochem. Biophys. Res. Comm. 150, 1155–61.

    PubMed  Google Scholar 

  • Labeit, S., Barlow, D. P., Gautel, M., Gibson, T., Holt, J., Hsieh, C. L., Francke, U., Leonard, K., Wardale, J., Whiting, A. &Trinick, J. (1990) A regular pattern of two types of 100-residue motif in the sequence of titin.Nature 345, 273–6.

    PubMed  Google Scholar 

  • Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage-T4.Nature 227, 680–5.

    PubMed  Google Scholar 

  • Maruyama, K. (1986) Connectin, an elastic filamentous protein of striated muscle.Int. Rev. Cytol. 104, 81–114.

    PubMed  Google Scholar 

  • Maruyama, K., Kimura, S., Yoshidomi, H., Sawada, H. &Kikuchi, M. (1984) Molecular size and shape ofβ-connectin, an elastic protein of striated muscle.J. Biochem. 95, 1423–33.

    PubMed  Google Scholar 

  • Maruyama, K., Yoshioka, T., Higuchi, H., Ohashi, K., Kimura, S. &Natori, R. (1985) Connectin filaments link thick filaments and Z-lines in frog skeletal muscle as revealed by immunoelectron mycroscopy.J. Cell Biol. 101, 2167–72.

    PubMed  Google Scholar 

  • Maruyama, K., Itoh, Y. &Arisaka, F. (1986) Circular dichroism spectra show abundance ofβ-sheet structure in connectin, a muscle elastic protein.FEBS Lett. 202, 353–5.

    PubMed  Google Scholar 

  • Maruyama, K., Hu, D. H., Suzuki, T. &Kimura, S. (1987) Binding of actin filaments to connectin.J. Biochem. 101, 1339–46.

    PubMed  Google Scholar 

  • Maruyama, K., Matsuno, A., Higuchi, H., Shimaoka, S., Kimura, S. &Shimizu, T. (1989) Behaviour of connectin (titin) and nebulin in skinned muscle fibres released after extreme stretch as revealed by immunoelectron microscopy.J. Muscle Res. Cell Motil. 10, 350–9.

    PubMed  Google Scholar 

  • Murayama, T., Nakauchi, Y., Kimura, S. &Maruyama, K. (1989) Binding of connectin to myosin filaments.J. Biochem. 105, 323–6.

    PubMed  Google Scholar 

  • Matsuno, A., Takano-Ohmuro, H, Itoh, Y., Matsuura, T., Shibata, M., Nakae, H., Kaminuma, T. &Maruyama, K. (1989) Anti-connectin monoclonal antibodies that react with unc-22 gene product bind dense bodies ofCaenorhabditis (nematode) bodywall muscle cells.Tissue & Cell 21, 537–44.

    Google Scholar 

  • Nave, R., Fürst, D. O. &Weber, K. (1989) Visualization of the polarity of isolated titin molecules: a single globular head on a long thin rod as the M band anchoring domain?J. Cell Biol. 109, 2177–87.

    PubMed  Google Scholar 

  • Perry, S. V. (1953) Preparation of myosin.Methods Enzymol.2, 582–8.

    Google Scholar 

  • Pierobon-Bormioli, S., Betto, R. &Salviati, G. (1989) The organization of titin (connectin) and nebulin in the sarcomeres: an immunocytolocalization study.J. Muscle Res. Cell Motil. 10, 446–56.

    PubMed  Google Scholar 

  • Provencher, S. W. &Glöckner, J. (1981) Estimation of globular protein secondary structure from circular dichroism.Biochemistry 20,33–7.

    PubMed  Google Scholar 

  • Salviati, G., Betto, R., Ceoldo, S. &Pierobon-Bormioli (1990) Morphological and functional characterization of the endosarcomeric elastic filament.Am. J. Physiol. 259 (Cell Physiol. 28), C144-C149.

    PubMed  Google Scholar 

  • Sonoda, M., Kimura, S., Moriya, H., Shimada, Y. &Maruyama, K. (1990) Molecular shape ofα-connectin, an elastic filamentous protein of skeletal muscle.Proc. Jap. Acad. 66B, 213–16.

    Google Scholar 

  • Spudich, J. A. &Watt, S. J. (1971) The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin.J. Biol. Chem. 246, 4866–71.

    Google Scholar 

  • Takahashi, K. &Takai, H. (1988) Transformation ofα-connectin toβ-connectin in rabbit skeletal muscle stored at 5°C.Abstract of the 80th Jap. Soc. Zootech. Sci. p. 102 (in Japanese).

  • Takeda, K. &Moriyama, Y. (1989) Secondary structural changes in the intact and the disulfide bridges cleavedβ-lactoglobulin A and B in solutions of urea, guanidine hydrochloride, and sodium dodecyl sulfate.J. Protein Chem. 8, 487–94.

    PubMed  Google Scholar 

  • Trinick, J. (1981) End-filaments: a new structural element of vertebrate skeletal muscle thick filaments.J. Mol. Biol. 151, 309–14.

    PubMed  Google Scholar 

  • Trinick, J., Knight, P. &Whiting, A. (1984) Purification and properties of native titin.J. Mol. Biol. 180, 331–56.

    PubMed  Google Scholar 

  • Wang, K., Ramirez-Mitchell, R. &Palter, D. (1984) Titin is an extraordinarily long, flexible, and slender myofibrillar protein.Proc. Natl. Acad. Sci. (USA) 81, 3685–9.

    Google Scholar 

  • Weber, K. &Osborn, M. (1969) The reliability of molecular weight determination by dodecyl sulphate-polyacrylamide gel electrophoresis.J. Biol. Chem. 244, 4406–12.

    PubMed  Google Scholar 

  • Whiting, A., Wardale, J. &Trinick, J. (1989) Does titin regulate the length of muscle thick filaments?J. Mol. Biol. 205, 263–8.

    PubMed  Google Scholar 

  • Williams, A. F. (1987) A year in the life of the immunoglobulin superfamily.Immunology Today 8, 299–303.

    Google Scholar 

  • Wu, C. S. C., Hasegawa, J., Smith, A. P., Loh, H. H., Lee, N. M. &Yang, J. T. (1990) Opioid-binding protein (OBCAM) is rich inβ-sheets.J. Protein Chem. 9, 3–7.

    PubMed  Google Scholar 

  • Yoshioka, T., Higuchi, H., Kimura, S., Ohashi, K., Umazume, Y. &Maruyama, K. (1986) Effects of mild trypsin treatment on the passive tension generation and connectin splitting in stretched skinned fibers from frog skeletal muscle.Biomed. Res. 7, 181–6.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biology, Faculty of Science, Chiba University, 260, Chiba, Japan

    Sumiko Kimura, Tetsu Matsuura, Satoshi Ohtsuka, Yuni Nakauchi & Koscak Maruyama

  2. Department of Biology, Facuity of Science, Shimane University, 690, Matsue, Japan

    Akira Matsuno

Authors
  1. Sumiko Kimura
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tetsu Matsuura
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Satoshi Ohtsuka
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yuni Nakauchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Akira Matsuno
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Koscak Maruyama
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kimura, S., Matsuura, T., Ohtsuka, S. et al. Characterization and localization of α-connectin (titin 1): An elastic protein isolated from rabbit skeletal muscle. J Muscle Res Cell Motil 13, 39–47 (1992). https://doi.org/10.1007/BF01738426

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01738426

Keywords

Search

Navigation