|Budget Amount *help
¥7,000,000 (Direct Cost : ¥7,000,000)
Fiscal Year 1991 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1990 : ¥1,100,000 (Direct Cost : ¥1,100,000)
Fiscal Year 1989 : ¥5,100,000 (Direct Cost : ¥5,100,000)
The giant muscle elastic protein, alpha-connectin, also called titin 1, of MW of about 3000 k was not isolated because of its insolubility and easy proteolysis. However, we were able to solubilize it with 0.2 M phosphate buffer of pH 7.0. Protease inhuibitor, leupeptin, lmM, hasd to be added. Fortunately, contaminant beta-connectin (MW, 2000 k) was not adsorbed to Toyopearl (DEAE) column in the presence of 4M urea. alpha-connection was eluted by 0.2 M NaCl.
When beta-connectin was split off from alpha-connection institue 1200 kDa peptide was formed. It was possible to isolate this peptide by sedimentation of alpha- and beta-connectin at low ionic strength. We prepared an antiserum against 1200kDa peptide (Pc (1200) ). This antibody reacted with alpha-connection but not with beta-connection, Immunoelectron microscopy revealed that Pc (1200) bound the Z line and the N_2 line region in the I band. The binding site in the I band was movable depending on sarcomere length. It is clear that the 1200kDa peptide covers the mother molecule, alpha-connection, from the Z line to the N_2 line region in the I band. beta-Connection covers it form the N_2 line region through a myosin filament up to the edge of the M line. A monoclonal antibody SMl, with does not react wilth 1200kDa peptide, also bound the region near the N_2 line.
Circular dichroism spectra suggested that alpha- and beta-connection and 1200kDa peptide all consisted of about 60% beta sheet and 30% beta turn. Ultraviolet resonance Raman spectra of beta-connection also indicated the abundance of beta sheet. Infrared dichroism spectra of oriented beta-connection fibers showed that beta sheet structure run parallel to the fiber axix. Therefore, beta sheet spiral stucture was denied.