Isolation and Characterization of Connection, Muscle Elastic Protein

巨型肌肉弹性蛋白α-Connectin，又称titin-1，分子量约为3000k，因其不溶性和易蛋白分解而未被分离到。然而，我们能够用0.2M的pH 7.0的磷酸盐缓冲液来溶解它。蛋白水解酶Inhuibitor，LeuPeptin，LMM，必须加入。幸运的是，在4M尿素存在下，污染物β-连接素(MW，2000k)没有被吸附在Toyota opearl(DEAE)柱上。用0.2M氯化钠洗脱α-连接素，将β-连接素从α-连接素中分离出来，形成1200 kDa的多肽。在低离子强度下，通过沉淀α-和β-连接素来分离这种多肽是可能的。制备了抗1200 kDa多肽的抗血清(Pc(1200))。免疫电子显微镜显示Pc(1200)结合在I带的Z线和N_2线区。根据肌节的长度，I带的结合部位是可移动的。很明显，1200 kDa的多肽覆盖了母体分子α-连接，从Z线到I带的N_2线区域。β-连接通过肌球蛋白细丝覆盖N_2线区域，直至M线的边缘。一种与1200 kDa多肽不反应的单抗SML也结合了N_2线附近的区域，圆二色谱表明，α-和β-连接和1200 kDa多肽均由约60%的β折叠和30%的β转折组成。β-连接的紫外共振拉曼光谱也表明了β-折叠的丰度。取向贝塔连接纤维的红外二向色性光谱表明，贝塔片层结构平行于纤维轴线。因此，贝塔片状螺旋结构被否定。

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.