Cell-free translation and partial characterization of mRNA coding for enkephalin-precursor protein.
The biosynthesis of the protein precursor of [Met]enkephalin (Tyr-Gly-Gly-Phe-Met) was studied using cell-free translation systems to characterize the enkephalin-precursor gene product and mRNA. Proteins obtained by translation of bovine adrenal medullary mRNA in the presence of [35S]methionine were digested with trypsin and carboxypeptidase B. The resultant peptides were immunoprecipitated with anti[Met]enkephalin serum and subsequently analyzed by reverse phase HPLC. [35S][Met]Enkephalin (up to 0.2 fmol/micrograms mRNA, identified by antigenic specificity and chromatographic mobility, was isolated from peptides obtained from proteins whose synthesis was dependent upon adrenal medullary mRNA. Both trypsin and carboxy-peptidase B were required to generate [35S][Met]enkephalin from translation products. The largest protein containing the [35S]-[Met]enkephalin sequence synthesized in the wheat germ system has a Mr, of 31,000 +/- 1000, determined by NaDodSO4/polyacrylamide gel electrophoresis. Adrenal medullary mRNA coding for protein containing the [35S][Met]enkephalin sequence was resolved into a major fraction of 1450 +/- 150 nucleotides and a minor fraction of 47000 +/- 450 nucleotides, as determined by agarose gel electrophoresis in the presence of methylmercuric hydroxide. It is proposed that the major enkephalin-precursor gene product of adrenal medulla is a protein of Mr approximately 31,000.U
D000313:Adrenal Medulla; D000818:Animals; D001483:Base Sequence; D043424:Carboxypeptidase B; D002268:Carboxypeptidases; D002417:Cattle; D002474:Cell-Free System; D004723:Endorphins / Q000235:genetics*; D004744:Enkephalin, Methionine; D004745:Enkephalins / Q000096:biosynthesis / Q000235:genetics*; D008970:Molecular Weight; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D014357:Trypsin
D004723:Endorphins; D004745:Enkephalins; D011498:Protein Precursors; D012333:RNA, Messenger; D004744:Enkephalin, Methionine; D002268:Carboxypeptidases; D043424:Carboxypeptidase B; D014357:Trypsin