Isolation and Characterization of Connection, Muscle Elastic Protein

巨肌弹性蛋白α -连接蛋白，又称titin 1，分子量约为3000 k，因其不溶性和易蛋白水解而未被分离。然而，我们可以用pH 7.0的0.2 M磷酸盐缓冲液将其溶解。蛋白酶抑制剂，白细胞介素，lmM，必须添加。幸运的是，在4M尿素存在的情况下，污染物β -连接蛋白（MW, 2000 k）没有被吸附到Toyopearl （DEAE）柱上。α -连接用0.2 M NaCl洗脱。连接蛋白与连接蛋白分离形成1200 kDa肽。在低离子强度下通过α -连接素和β -连接素的沉淀分离这种肽是可能的。我们制备了抗1200kDa肽的血清（Pc(1200)）。该抗体与α -连接发生反应，但与β -连接不发生反应。免疫电镜显示，Pc（1200）结合了I波段的Z线和N_2线区域。I带的结合位点随肌节长度的变化而变化。很明显，1200kDa肽覆盖了I波段从Z线到N_2线的母分子α -连接。连接从n2线区域通过肌凝蛋白丝一直覆盖到M线边缘。与1200kDa肽不发生反应的单克隆抗体SMl也结合在N_2线附近。圆二色光谱表明，α -和β -连接以及1200kDa肽均由约60%的β片和30%的β转组成。β连接的紫外共振拉曼光谱也显示了β片的丰度。定向β连接纤维的红外二色性光谱表明，β片结构平行于纤维轴。因此，否定了β片的螺旋结构。

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.