Calcium binding to an elastic portion of connectin/titin filaments

R. Tatsumi, K. Maeda, A. Hattori, K. Takahashi

Research output: Contribution to journalArticle

67 Citations (Scopus)

Abstract

α-Connectin/titin-1 exists as an elastic filament that links a thick filament with the Z-disk, keeping thick filaments centered within the sarcomere during force generation. We have shown that the connectin filament has an affinity for calcium ions and its binding site(s) is restricted to the β-connectin/titin-2 portion. We now report the localization and the characterization of calcium-binding sites on β-connectin. Purified β-connectin was digested by trypsin into 1700- and 400-kDa fragments, which were then subjected to fluorescence calcium-binding assays. The 400-kDa fragment possesses calcium-binding activity; the binding constant was 1.0 × 107 M-1 and the molar ratio of bound calcium ions to the 400-kDa fragment reached a maximum of 12 at a free calcium ion concentration of approximately 1.0 μM. Antibodies against the 400-kDa fragment formed a sharp dense stripe at the boundary of the A and the I bands, indicating that the calcium-binding domain constitutes the N-terminal region of β-connectin, that is, the elastic portion of connectin filaments. Furthermore, we estimated the N-terminal location of β-connectin of various origins (n = 26). Myofibrils were treated with a solution containing 0.1 mM CaCl2 and 70 μM leupeptin to split connectin filaments into β-connectin and a subfragment, and chain weights of these polypeptides were estimated according to their mobility in 2% polyacrylamide slab gels. The subfragment exhibited a similar chain weight of 1200 ± 33 kDa (mean ± SD), while α- and β-connectins were variable in size according to their origin. These results suggest that the apparent length of the 1200-kDa subfragment portion is almost constant in all instances, about 0.34 μm at the slack condition, therefore that the C-terminus of the 1200-kDa subfragment, that is, the N-terminus of the calcium-binding domain, is at the N2 line region of parent filaments in situ. Because the secondary structure of the 400-kDa fragment was changed by the binding of calcium ions, connectin filaments could be expected to alter their elasticity during the contraction-relaxation cycle of skeletal muscle.

Original languageEnglish
Pages (from-to)149-162
Number of pages14
JournalJournal of Muscle Research and Cell Motility
Volume22
Issue number2
DOIs
Publication statusPublished - Aug 20 2001
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Physiology
  • Cell Biology

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