Cell-free biosynthesis of multiple preprosomatostatins: characterization by hybrid selection and amino-terminal sequencing.
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.
D000595:Amino Acid Sequence; D000818:Animals; D002474:Cell-Free System; D005399:Fishes; D007425:Intracellular Membranes / Q000378:metabolism; D007515:Islets of Langerhans / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D010446:Peptide Fragments / Q000032:analysis; D010944:Plants / Q000378:metabolism; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D013004:Somatostatin / Q000031:analogs & derivatives* / Q000235:genetics; D014908:Triticum / Q000378:metabolism; D014357:Trypsin
D010446:Peptide Fragments; D011498:Protein Precursors; D012333:RNA, Messenger; D013004:Somatostatin; D014357:Trypsin