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
.