CELL-FREE BIOSYNTHESIS OF MULTIPLE PREPROSOMATOSTATINS - CHARACTERIZATION BY HYBRID SELECTION AND AMINO-TERMINAL SEQUENCING
CELL-FREE BIOSYNTHESIS OF MULTIPLE PREPROSOMATOSTATINS - CHARACTERIZATION BY HYBRID SELECTION AND AMINO-TERMINAL SEQUENCING
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DOI：
10.1021/bi00307a023
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发表时间：
1984-01-01
期刊：
影响因子：
2.9
通讯作者：
SHIELDS, D
中科院分区：
文献类型：
--
作者：
WARREN, TG;SHIELDS, D
从 [Lophius americanus] Langerhans 胰岛分离的 mRNA 进行体外翻译，可合成 MW 19,000、18,000 和 16,000 的 3 种主要前促生长素抑制素，每种都可以分解为几种等电形式。生长抑素前体的进一步表征通过特定前生长抑素 mRNA 的杂交选择翻译、由杂交选择的 mRNA 合成的新生前生长抑素的体外蛋白水解加工、其胰蛋白酶肽的比较以及信号肽区域的部分氨基末端序列分析来呈现。使用特定c[互补]DNA克隆的杂交选择实验证明了前促生长素抑制素种类对应于先前表征的前体cDNA；因此，由质粒pLaS1编码的多肽对应于MW 18,000前促生长素抑制素的一种形式，而MW 16,000前促生长素抑制素的一种形式由pLaS2编码。对胰蛋白酶肽的分析表明，MW 16,000 分子在羧基末端具有成熟的激素序列，正如 MW 19,000 和 18,000 前体所显示的那样。部分NH2末端序列分析证实了杂交选择的数据，并证明MW 18,000前体含有信号肽，在每个前促生长素抑制素信号肽的某些位置处表现出氨基酸异质性。这种异质性可能部分解释了前促生长素抑制素分子的变异。
In vitro translation of mRNA isolated from [Lophius americanus] islets of Langerhans results in the synthesis of 3 major preprosomatostatins of MW 19,000, 18,000 and 16,000, each of which can be resolved into several isoelectric forms. Further characterization of the somatostatin precursors is presented by hybrid selection translation of specific preprosomatostatin mRNA, in vitro proteolytic processing of the nascent preprosomatostatins synthesized from hybrid-selected mRNA, comparison of their tryptic peptides and partial amino-terminal sequence analysis of the signal peptide regions. Hybrid selection experiments using specific c[complementary]DNA clones demonstrated which preprosomatostatin species corresponded to previously characterized precursor cDNA; thus, the polypeptide encoded by plasmid pLaS1 corresponds to one form of the MW 18,000 preprosomatostatin while one form of the MW 16,000 preprosomatostatin is encoded by pLaS2. Analysis of the tryptic peptides demonstrated that the MW 16,000 molecule possessed the mature hormone sequence at the carboxyl terminus, as had been shown for the MW 19,000 and 18,000 precursors. Partial NH2-terminal sequence analysis confirmed the data from hybrid selection and demonstrated that the MW 18,000 precursor contained a signal peptide manifesting amino acid heterogeneity at certain positions in the signal peptides of each preprosomatostatin. This heterogeneity might account, in part, for variants of the preprosomatostatin molecules.