Tan K O, Sater G R, Myers A M, Robson R M, Huiatt T W
Department of Animal Science, Iowa State University, Ames 50011.
J Biol Chem. 1993 Oct 25;268(30):22900-7.
Titin is an approximately 3000-kDa polypeptide that constitutes a set of elastic filaments that connect thick filaments to the Z-line in vertebrate striated muscle myofibrils. To characterize the primary structure of titin, three overlapping cDNA clones comprising 2.4 kilobases of avian muscle titin coding sequence were obtained from a cDNA library constructed from embryonic chick cardiac muscle RNA size-selected for large transcripts. Expression of one cDNA clone in Escherichia coli produced a fusion protein that reacted specifically with titin antibodies, and titin antiserum affinity-purified against this fusion protein reacted specifically with titin on immunoblots of chicken cardiac and skeletal muscle myofibrils. Indirect immunofluorescence localization with the fusion protein-specific antibodies demonstrated that the cDNA sequence was from the region of titin located in the myofibrillar A-band adjacent to the A/I junction. Derived amino acid sequences demonstrated a repeating pattern of fibronectin type III and immunoglobulin C2 motifs, as shown previously for a portion of rabbit skeletal muscle titin located in the central region of the A-band and for other myofibrillar proteins that bind to myosin. Differences between the rabbit and chicken titin sequences included a unique, serine-rich region in one motif, which represents a potential phosphorylation site. This is the first report of sequence information for avian titin from a previously uncharacterized portion of the titin molecule.
肌联蛋白是一种分子量约为3000 kDa的多肽，它构成了一组弹性细丝，在脊椎动物横纹肌肌原纤维中将粗肌丝连接到Z线。为了表征肌联蛋白的一级结构，从一个由胚胎鸡心肌RNA构建的cDNA文库中获得了三个重叠的cDNA克隆，这些克隆包含2.4千碱基的禽肌肌联蛋白编码序列，该文库对大转录本进行了大小选择。一个cDNA克隆在大肠杆菌中的表达产生了一种与肌联蛋白抗体特异性反应的融合蛋白，用这种融合蛋白亲和纯化的肌联蛋白抗血清在鸡心肌和骨骼肌肌原纤维的免疫印迹上与肌联蛋白特异性反应。用融合蛋白特异性抗体进行间接免疫荧光定位表明，该cDNA序列来自肌联蛋白位于肌原纤维A带中靠近A/I交界处的区域。推导的氨基酸序列显示出纤连蛋白III型和免疫球蛋白C2基序的重复模式，如先前在位于A带中心区域的兔骨骼肌肌联蛋白的一部分以及其他与肌球蛋白结合的肌原纤维蛋白中所示。兔和鸡肌联蛋白序列之间的差异包括一个基序中独特的富含丝氨酸的区域，这代表一个潜在的磷酸化位点。这是关于肌联蛋白分子中一个以前未表征部分的禽肌联蛋白序列信息的首次报道。
