Immunological identification of a common precursor to arginine vasopressin and neurophysin II synthesized by in vitro translation of bovine hypothalamic mRNA.

Abstract

mRNA from membrane-bound polysomes of bovine hypothalamus was translated in an mRNA-dependent cellfree system from reticulocyte lysate or wheat germ. The translation products were identified by immunoprecipitation with antibodies to either neurophysin H or arginine vasopressin followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. An immunoreactive polypeptide was obtained with an apparent Mr of 21,000. Sequential immunoprecipitation studies indicated that the Mr 21,000 product is a common precursor to neurophysin II and arginine vasopressin. The specificity of the immunoprecipitation was demonstrated by competition with excess amounts of unlabeled neurophysin H or arginine vasopressin; little or no competition was observed with unlabeled neurophysin I or oxytocin. Processing experiments with microsomal membranes from dog pancreas or tunicamycin-treated ascites tumor cells showed that the Mr 21,000 polypeptide is the prepro form. It was converted to a pro form with Mr 19,000 suggesting a pre sequence of approximately 15 amino acids. The Mr 19,000 polypeptide was coreglycosylated to an apparent Mr of 23,000, indicating that the neurophysin II-arginine vasopressin precursor is a glycopolypeptide.Oligopeptides such as the bacterial antibiotics gramicidin and tyrocidin are synthesized in a nonribosomal fashion on polyenzyme templates (1, 2). Reports that oligopeptides of hypothalamus from mammals also might be synthesized by a nonribosomal mechanism are still controversial (3-5). In vitro biosynthesis of the hypothalamic nonapeptide hormones vasopressin and oxytocin has not yet been resolved. Although Sachs (6) suggested more than a decade ago that vasopressin may be synthesized by a ribosome-dependent mechanism via a precursor polypeptide, only recently a tentative glycopolypeptide precursor with an apparent M, of 20,000 has been isolated from the hypothalamus of rats and mice (7-9). This precursor gave rise to a vasopressin-like peptide after trypsinization (7). In addition the precursor crossreacted with antibodies raised against one of the hypothalamic cysteine-rich neurophysins (8,9) which is known to transport vasopressin to its storage site in the posterior pituitary (10).If the precursor hypothesis is valid it should be possible to demonstrate biosynthesis of a vasopressin-neurophysin precursor directly by translation of hypothalamic mRNA in cellfree systems. Although a number of groups have reported the synthesis of neurophysin precursors in cell-free systems (11-13), identification of vasopressin-like sequences within the reported precursors has been lacking. The present communication reports mRNA-directed synthesis of a common precursor to arginine vasopressin (AVP) and neurophysin II and its processing and core glycosylation; specific antibodies were used for identification. MATERIALS AND METHODSBovine neurophysin I (Np I) and neurophysin II (Np II) and rabbit antisera to Np I and Np II were purchased from Bioproducts (Brussels); according to the supplier the antisera against Np ...

Arginine Vasopressin (AVP) and AVP-Like Immunoreactivity in Peripheral Tissues

“…1). This had been suggested in earlier biosynthetic labeling (63)(64)(65)(66) and also in cell-free translation studies (67,68). Similarly, the bovine hypothalamic cDNA nucleotide sequence for the OT-NPI precursor (69) OT and NPI are derived from a single, but shortened, precursor hormone since the C-terminal glycopeptide moiety is missing (Fig.…”

Copeptin Immunoreactivity and Calcium Mobilisation in Hypothalamic Neurones of the Rat

“…Vasopressin prohormone is processed in mammalian cells to three fragments: VP, neurophysin II and a glycoprotein of 39 residues, except the guinea pig which has 38 residues (19), referred to as copeptin (20–23); the processing is complete at the level of posterior pituitary (10). In addition to being secreted conjointly into the portal system, vasopressin and copeptin may be liberated synaptically from vasopressin‐containing neurones to several well defined groups of neurones in the hindbrain and spinal cord (24).…”

The contribution of recombinant DNA techniques to reproductive biology

“…At the NH2-termini of both precursors are signal peptides thought to be involved in the translocation of prohormones into endoplasmic reticulum for subsequent transport to the Golgi apparatus. During this translocation the signal peptides are removed proteolytically (Austen, 1979;Kreil, 1981;Schmale & Richter, 1981). Cleavage seems to take place at an alanine residue and thereby conforms with over 50% of other known signal peptide cleavage sites (Austen, 1979).…”