Calcium binding to an elastic portion of connectin/titin filaments

Citation
R. Tatsumi et al., Calcium binding to an elastic portion of connectin/titin filaments, J MUSCLE R, 22(2), 2001, pp. 149-162
Citations number
86
Categorie Soggetti
Cell & Developmental Biology
Journal title
JOURNAL OF MUSCLE RESEARCH AND CELL MOTILITY
ISSN journal
01424319 → ACNP
Volume
22
Issue
2
Year of publication
2001
Pages
149 - 162
Database
ISI
SICI code
0142-4319(2001)22:2<149:CBTAEP>2.0.ZU;2-H
Abstract
alpha -Connectin/titin-1 exists as an elastic filament that links a thick f ilament with the Z-disk, keeping thick filaments centered within the sarcom ere 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 b eta -connectin/titin-2 portion. We now report the localization and the char acterization of calcium-binding sites on beta -connectin. Purified beta -co nnectin was digested by trypsin into 1700- and 400-kDa fragments, which wer e then subjected to fluorescence calcium-binding assays. The 400-kDa fragme nt possesses calcium-binding activity; the binding constant was 1.0 x 10(7) M-1 and the molar ratio of bound calcium ions to the 400-kDa fragment reac hed a maximum of 12 at a free calcium ion concentration of approximately 1. 0 muM. 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-bindi ng domain constitutes the N-terminal region of beta -connectin, that is, th e elastic portion of connectin filaments. Furthermore, we estimated the N-t erminal location of beta -connectin of various origins (n = 26). Myofibrils were treated with a solution containing 0.1 mM CaCl2 and 70 muM leupeptin to split connectin filaments into beta -connectin and a subfragment, and ch ain weights of these polypeptides were estimated according to their mobilit y in 2% polyacrylamide slab gels. The subfragment exhibited a similar chain weight of 1200 +/- 33 kDa (mean +/- SD), while alpha- and beta -connectins were variable in size according to their origin. These results suggest tha t the apparent length of the 1200-kDa subfragment portion is almost constan t in all instances, about 0.34 mum at the slack condition, therefore that t he C-terminus of the 1200-kDa subfragment, that is, the N-terminus of the c alcium-binding domain, is at the N-2 line region of parent filaments in sit u. Because the secondary structure of the 400-kDa fragment was changed by t he binding of calcium ions, connectin filaments could be expected to alter their elasticity during the contraction-relaxation cycle of skeletal muscle .