Calcium binding to an elastic portion of connectin/titin filaments.

摘要: Alpha-connectin/titin-1 exists as an elastic filament that links a thick with the Z-disk, keeping filaments centered within sarcomere during force generation. We have shown connectin has affinity for calcium ions and its binding site(s) is restricted to beta-connectin/titin-2 portion. now report localization characterization of calcium-binding sites on beta-connectin. Purified beta-connectin was digested by trypsin into 1700- 400-kDa fragments. which were then subjected fluorescence assays. The fragment possesses activity; constant 1.0 x 10(7) M(-1) molar ratio bound reached maximum 12 at free ion concentration approximately microM. Antibodies against formed sharp dense stripe boundary A I bands, indicating domain constitutes N-terminal region beta-connectin, is, portion filaments. Furthermore, we estimated location various origins (n = 26). Myofibrils treated solution containing 0.1 mM CaCl2 70 microM leupeptin split subfragment, chain weights these polypeptides according their mobility in 2% polyacrylamide slab gels. subfragment exhibited similar weight 1200+/-33 kDa (mean+/-SD), while alpha- beta-connectins variable size origin. These results suggest apparent length 1200-kDa almost all instances, about 0.34 microm slack condition, therefore C-terminus N-terminus domain, N2 line parent situ. Because secondary structure changed ions, could be expected alter elasticity contraction-relaxation cycle skeletal muscle.