Warren T G, Shields D
Biochemistry. 1984 Jun 5;23(12):2684-90. doi: 10.1021/bi00307a023.
In vitro translation of mRNA isolated from islets of Langerhans results in the synthesis of three major preprosomatostatins of Mr 19 000, 18 000, and 16 000, each of which can be resolved into several isoelectric forms [Warren, T. G., & Shields, D. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3729-3733]. Here we present further characterization of the somatostatin precursors by (i) hybrid selection translation of specific preprosomatostatin mRNAs, (ii) in vitro proteolytic processing of the nascent preprosomatostatins synthesized from hybrid-selected mRNAs, (iii) comparison of their tryptic peptides, and (iv) partial amino-terminal sequence analysis of the signal peptide regions. Hybrid selection experiments using specific cDNA clones demonstrated which preprosomatostatin species corresponded to previously characterized precursor cDNAs [Hobart, P., Crawford, R., Shen, L. P., Picket, R., & Rutter, W. J. (1980) Nature (London) 288, 137-141]; thus, the polypeptide encoded by plasmid pLaS1 corresponds to one form of the Mr 18 000 preprosomatostatins while one form of the Mr 16 000 preprosomatostatins is encoded by pLaS2. Analysis of the tryptic peptides demonstrated that the Mr 16 000 molecule possessed the mature hormone sequence at the carboxyl terminus, as had been shown for the Mr 19 000 and 18 000 precursors. Partial NH2-terminal sequence analysis (a) confirmed the data from hybrid selection and (b) demonstrated that the Mr 18 000 precursor contained a signal peptide manifesting amino acid heterogeneity at certain positions in the signal peptides of each preprosomatostatin. It is suggested that this heterogeneity might account, in part, for variants of the preprosomatostatin molecules.
从胰岛分离的mRNA的体外翻译产生了三种主要的前胰岛素原生长抑素，分子量分别为19000、18000和16000，每种都可分解为几种等电形式[沃伦，T.G.，& 希尔兹，D.（1982年）美国国家科学院院刊79，3729 - 3733]。在此，我们通过以下方式对生长抑素前体进行进一步表征：（i）特定前胰岛素原生长抑素mRNA的杂交选择翻译，（ii）由杂交选择的mRNA合成的新生前胰岛素原生长抑素的体外蛋白水解加工，（iii）它们的胰蛋白酶肽段比较，以及（iv）信号肽区域的部分氨基末端序列分析。使用特定cDNA克隆的杂交选择实验证明了哪些前胰岛素原生长抑素物种对应于先前表征的前体cDNA [霍巴特，P.，克劳福德，R.，沈，L.P.，皮克特，R.，& 鲁特，W.J.（1980年）自然（伦敦）288，137 - 141]；因此，质粒pLaS1编码的多肽对应于分子量为18000的前胰岛素原生长抑素的一种形式，而分子量为16000的前胰岛素原生长抑素的一种形式由pLaS2编码。胰蛋白酶肽段分析表明，分子量为16000的分子在羧基末端具有成熟激素序列，这与分子量为19000和18000的前体情况相同。部分氨基末端序列分析（a）证实了杂交选择的数据，并且（b）表明分子量为18,000的前体包含一个信号肽，在每个前胰岛素原生长抑素的信号肽的某些位置表现出氨基酸异质性。有人认为这种异质性可能部分解释了前胰岛素原生长抑素分子的变体。
