MacGillivray R T, Davie E W
Biochemistry. 1984 Apr 10;23(8):1626-34. doi: 10.1021/bi00303a007.
Prothrombin mRNA has been enriched 20-60-fold by using specific immunoadsorption of bovine liver polysomes. The enriched mRNA was translated in a cell-free protein synthesizing system derived from rabbit reticulocytes, and the radiolabeled translation product was isolated by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The radiolabeled prothrombin synthesized in the cell-free system was then subjected to automated Edman degradation and shown to contain a leader sequence of at least 30 residues that was rich in leucine, phenylalanine, and alanine. In order to fully characterize the leader sequence for prothrombin, a bovine liver cDNA library was constructed containing DNA inserts of over 1000 base pairs. Two cDNA clones coding for bovine prothrombin were isolated from the library and their nucleotide sequences determined. A leader sequence of 43 amino acids was predicted from the sequence of the cDNA, and the first 30 residues were in agreement with the partial sequence obtained by the cell-free protein synthesizing system. From the amino acid sequence of the leader sequence, it is proposed that bovine prothrombin is synthesized with a prepro leader sequence starting with a methionine residue at position -43. The amino acid sequence of the mature prothrombin molecule circulating in plasma was also predicted from the cDNA and shown to be in good agreement with that determined previously by conventional amino acid sequence analysis.
通过对牛肝多核糖体进行特异性免疫吸附，凝血酶原mRNA已富集了20至60倍。将富集的mRNA在源自兔网织红细胞的无细胞蛋白质合成系统中进行翻译，然后通过免疫沉淀和十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳分离放射性标记的翻译产物。在无细胞系统中合成的放射性标记凝血酶原随后进行自动埃德曼降解，结果显示其含有至少30个残基的前导序列，该序列富含亮氨酸、苯丙氨酸和丙氨酸。为了全面表征凝血酶原的前导序列，构建了一个包含超过1000个碱基对DNA插入片段的牛肝cDNA文库。从该文库中分离出两个编码牛凝血酶原的cDNA克隆，并测定了它们的核苷酸序列。从cDNA序列预测出一个43个氨基酸的前导序列，前30个残基与通过无细胞蛋白质合成系统获得的部分序列一致。根据前导序列的氨基酸序列，推测牛凝血酶原是以一个从第 -43位的甲硫氨酸残基开始的前原前导序列进行合成的。还从cDNA预测了血浆中循环的成熟凝血酶原分子的氨基酸序列，结果显示与先前通过传统氨基酸序列分析确定的序列高度一致。
