title: Effect of neurophysin on enzymatic maturation of oxytocin from its precursor.
abstract: We examined the extent to which rates of enzymatic conversion of the oxytocin biosynthetic precursor to mature peptide are modulated by intramolecular and intermolecular assembly of precursor and polypeptide intermediates. The biosynthesized precursor contains hormone and neurophysin sequences linked by a Gly-Lys-Arg sequence and undergoes enzymatic processing reactions which include endoproteolytic cleavage at the Lys-Arg dibasic sequence, carboxypeptidase B-like exoproteolytic cleavage, and enzymatic amidation. We evaluated the effect of neurophysin on such processing reactions using semisynthetic precursors of oxytocin/bovine neurophysin I and synthetic oxytocinyl precursor intermediates as substrates. Neurophysin I at high concentration (0.7 mM) reduced the rates of carboxy-peptidase B-like conversion of oxytocinyl-Gly-Lys-Arg to oxytocinyl-Gly and the enzymatic amidation of oxytocinyl-Gly to mature (C-terminal amidated) oxytocin. The dependence of rate suppression on the concentrations of peptide substrate and neurophysin I suggested that suppression is due to intermolecular formation of hormone-neurophysin complexes which are aggregated at least to dimers. An analogous intramolecular neurophysin effect was found for endoproteolytic processing of semisynthetic precursors. Endoproteinase Lys-C cleaved the Lys11-Arg12 peptide bond in a native-like semisynthetic precursor at a significantly slower rate than it did an assembly-deficient precursor analogue. The difference in semisynthetic precursor endoproteolysis rates is most substantial at the high concentrations at which the native-like precursor would form dimers but the assembly-deficient analogue would not. The native-like semisynthetic precursor was more stable than the assembly-deficient precursor analogue to tryptic digestion. The concentration-dependent effects of neurophysin, both intramolecularly as a precursor domain and intermolecularly as an interacting protein, are likely to occur in the secretory granules in which the biosynthetic precursors are packaged. The molecular organization of both hormone/neurophysin precursors and the noncovalently complexed hormone-neurophysin intermediates can be expected to play a role in modulating enzymatic processing reactions that lead to mature neurohypophysial hormones.
authors.affiliation: Molecular, Cellular, and Nutritional Endocrinology Branch, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: S
authors.initials: S
authors.lastname: Ando
authors.forename: A S
authors.initials: AS
authors.lastname: Murthy
authors.forename: B A
authors.initials: BA
authors.lastname: Eipper
authors.forename: I M
authors.initials: IM
authors.lastname: Chaiken
mesh_terms: D000818:Animals; D043424:Carboxypeptidase B; D002268:Carboxypeptidases / Q000378:metabolism; D002417:Cattle; D002846:Chromatography, Affinity; D010450:Endopeptidases / Q000378:metabolism; D008666:Metalloendopeptidases; D009481:Neurophysins / Q000494:pharmacology*; D010121:Oxytocin / Q000031:analogs & derivatives* / Q000378:metabolism*; D013552:Swine; D014357:Trypsin / Q000378:metabolism
chemical_list: D009481:Neurophysins; D010121:Oxytocin; C046278:oxytocin, Gly-Lys-Arg-; D002268:Carboxypeptidases; D010450:Endopeptidases; D043424:Carboxypeptidase B; D014357:Trypsin; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase