Chemical Synthesis and Enzymic Processing of Precursor Forms of Cecropins A and B

作者：Hans G. Boman, I A Boman, David Andreu, Zhi‐Qing Li, Robert Bruce Merrifield, Gabriel Schlenstedt, Richard Zimmermann · 发表于：Journal of Biological Chemistry · 年份：1989 · DOI：10.1016/s0021-9258(18)83628-6 · 被引用次数：115 · 研究领域：Neuropeptides and Animal Physiology、Peptidase Inhibition and Analysis、Chemical Synthesis and Analysis

Radiolabeled preprocecropin B, with an alpha-amidated COOH terminus, and preprocecropin A, extended at the COOH terminus by a glycine residue, were synthesized by solid-phase methods. The respective syntheses were interrupted at intervals to allow the preparation of the predicted procecropins A and B as well as three other truncated derivatives of the cecropin A precursors. All the synthetic peptides were purified to near homogeneity by reverse-phase liquid chromatography and their purity was established by analytical high performance liquid chromatography, gel electrophoresis, and amino acid analysis. A dipeptidyl aminopeptidase was purified about 350 times from the hemolymph of cecropia pupae and characterized by its affinity for different substrates and inhibitors. The synthetic prepro peptides were tested for processing by an extract of dog pancreas microsomes and purified leader peptidase from Escherichia coli, with and without partly purified dipeptidyl aminopeptidase, and the two synthetic proforms were also processed with the dipeptidyl aminopeptidase alone. From these experiments we conclude that the signal/leader peptidase cleaves the peptide bond between Ala-5 and Ala-4. This cleavage site is further substantiated by radio sequencing of procecropin A isolated after synthesis in a coupled system for in vitro transcription, translation, and processing. The two procecropins, which are stable to further digestion by the signal peptidase, are further processed by dipept...