NEW ELASTIC STRUCTURE OF GIANT SARCOMERE

在脊椎动物横纹肌中，连接蛋白（也称为连接蛋白）（MM 3000 kDa）从粗丝上的Z线延伸到肌节的M线。一个分子在静止状态下横跨半个约1.2μm长的肌节。另一方面，在静止状态下长度为8.3μm的小龙虾的巨大肌节中，有一种类似大小的弹性蛋白起着连接作用。3000kda的蛋白质是如何覆盖这么长的距离的？利用从螯虾近缘cDNA文库中克隆的cDNA序列，预测了3000 kDa蛋白的全序列。与脊椎动物连接蛋白的序列有明显不同。首先，有68和71个氨基酸残基的新的重复基序。其次，在脊椎动物连接蛋白中丰富的免疫球蛋白C2基序或III型纤维连接蛋白的数量非常少。第三，有两个大的弹性PEVK域，而在脊椎动物连接中只有一个域。第四，肌球蛋白轻链激酶结构域缺失，与连接蛋白、投射蛋白和抽动蛋白共同。免疫荧光显微镜显示，当肌节被拉伸至约13μm时，68个残基重复序列和重复序列与免疫球蛋白结构域之间的间隔明显可扩展。此外，圆二色性光谱表明，新的68重复融合蛋白由53%的β-sheet和22%的β-turn组成。

In vertebrate striated muscle, connectin (also called titin ) (MM 3000 kDa) extends from the Z line over the thick filament up to the M line of the sarcomere. One molecule spans a half sarcomere approximately 1.2μm long at rest. On the other hand, in giant sarcomeres of crayfish claw closer muscle the length of which is 8.3μm long at rest, a similar size of an elastic protein functions as connetin. How does the 3000 kDa protein cover such a long distance?The entire sequence of the 3000 kDa protein was predicted from the cDNA sequences cloned from crayfish claw closer cDNA library. Markedly different sequence from that of vertebrate connectin has been found. First of all, there are novel repeating motifs of 68 and 71 amino acid residues. Second, the number of immunogloblin C2 motif or fibronectin Type III, abundant in vertebrate connectin, is very small. Third, there are two large elastic PEVK domains, as compared to only one domain in vertebrate connectin. Fourth, the myosin light chain kinase domain common to connectin, projectin and twitchin is missing. Immunofluorescence microscopy using antibodies raised to three recombinant peptides demonstrated that the 68 residue repeats and a long spacer between the repeats and immunoglobulin domains were remarkably extensible when the sarcomere was stretched to a sarcomere length of 〜13μm. Furthermore, circular dichroism spectra suggested that a fusion protein of novel 68 repeat consists of 53% β-sheet and 22% β-turn.