Fingerprint Analysis of Bovine Hypothalamic Preprosomatostatin

Abstract

Messenger RNA from bovine hypothalami was used to direct the synthesis in vitro of a precursor to somatostatin (SRIF) of Mr 15500. Specific antibodies, raised against the chemically synthesized tetradecapeptide SRIF‐14, were used for the preliminary characterzation. The radioactively labelled preprosomatostatin was then cleaved by trypsin or cyanogen bromide and the products were assayed by two‐dimensional fingerprinting techniques. The results conclusively demonstrated the presence of the tetradecapeptide SRIF‐14 sequence and its naturally occurring N‐terminally extended form, SRIF‐28. This 28‐amino‐acid sequence was shown to occupy the C terminus of the 15 500‐dalton precursor and is probably preceded by basic amino acid(s).

Human neuropeptide Y, somatostatin and vasopressin precursors identified in cell‐free translations of hypothalamic mRNA

“…This prepro-form is consistent with that predicted by the human gene sequence, where the somatostatin-28 molecule, and also the internal somatostatin-14 sequence, occupy the C-terminus of the precursor. It is thus also comparable to the hypothalamic preprosomatostatins recently described for other mammalian species [4,9].…”

“…All these antisera have been demonstrated to be highly specific for their respective 176 antigenic sequences. Identification of precursors from other mammals using these antisera has been confirmed by tryptic peptide mapping or subsequent sequence analysis [8,9]. Anti-neuropeptide Y was raised in rabbits using porcine neuropeptide Y (by courtesy of Dr K. Tatemoto) conjugated to bovine serum albumin using carbodiimide.…”

The gene for the hypothalamic peptide hormone oxytocin is highly expressed in the bovine corpus luteum: biosynthesis, structure and sequence analysis.

“…In vitro translation and immunoprecipitation of specific products Luteal or hypothalamic poly(A) I RNA was translated in a rabbit reticulocyte lysate system (New England Nuclear, Dreieich, FRG) using [35S]cysteine as radiolabel. 25 1u reaction mixtures were programmed with 0.5 Ag poly(A)+ RNA and immunoprecipitated using 30 Ag of IgG as described elsewhere (Schmale and Richter, 1981;Ivell and Richter, 1982). Control immunoprecipitations included unlabelled antigen (10-30 jg) as competing peptide.…”

“…Lanes 3 and 4: immnunospecific polypeptides selected by anti-NpI(UCB Bioproducts, Brussels) in the absence (lane 3), or presence (lane 4) of 10 Ag unlabelled NpI. Lanes 5 and 6: the NpI-immunoreactive polypeptides from 4 x 50 jAd translation reactions were eluted from an unfluorographed polyacrylamide gel(Ivell and Richter, 1982) and reacted with 30 ,ig antioxytocin (Ferring, Kiel, FRG) in the absence (lane 5) or presence (lane 6) of 30 Atg unlabelled oxytocin. Lane 7: as lane 3 except using anti-vasopressin IgG.…”