Immunological and chemical identification of a neurophysin-containing protein coded by messenger RNA from bovine hypothalamus

Abstract

The biosynthetic origin of the 10,000 MW neurophysins, carriers of the peptide hormones oxytocin and vasopressin, was studied by cell-free synthesis. Poly(A)-RNA was isolated from bovine hypothalamus and translated in a wheat germ system containing [35S]- or [3H]-labeled amino acids. A number of unique [35S]cysteine but few [35S]-Met-labeled proteins were coded by hypothalamic mRNA. A single, major, isotopically labeled protein (MW 23,000-25,000) was immunoprecipitated from these translation mixtures by addition of purified antibodies against bovine neurophysin II and subsequent addition of Cowan I strain of Staphylococcus aureus. Specificity of the immunoprecipitation was demonstrated by competition with unlabeled authentic neurophysins and the absence of competition with structurally unrelated ovalbumin. Neither nonimmune serum nor purified antibodies against ribonuclease immunoprecipitated the protein. The [35S]cysteine-labeled protein that was specifically immunoprecipitated was oxidized with performic acid and digested with trypsin in the presence of unlabeled, authentic bovine neurophysin II. Peptide mapping revealed that most of the major [35S]cysteine-labeled peptides (of the translation product) were identical to major cysteine-containing peptides of authentic neurophysin. Hypothalamic mRNA evidently directs the translation of several unique cysteine-rich proteins in an in vitro cell-free system. One of these proteins, which has a higher MW than authentic neurophysin, is recognized by purified antibodies to bovine neurophysin II and has cysteine-containing tryptic peptides in common with those of authentic neurophysin. This protein may be the primary translation product, pre-pro-neurophysin.