Pituitary enzyme conversion of putative synthetic oxytocin precursor intermediates.
Neurosecretory granule lysate from bovine posterior pituitary was shown to contain both carboxypeptidase B and amidating activities. The former sequentially releases COOH-terminal basic residues from the oxytocin biosynthetic precursor fragment oxytocinyl-GKR (CYIQNCPLGKR) to form oxytocinyl-GK and then oxytocinyl-G. The amidating enzyme converts the resulting oxytocinyl-G into oxytocin (CYIQNCPLG-NH2). The carboxypeptidase B was separated from a less specific carboxypeptidase present in granule lysate by gel filtration on Sephacryl S-300. Percoll density gradient centrifugation (after preliminary differential centrifugation) also yielded granule fractions enriched in the specific carboxypeptidase B activity. The carboxypeptidase B which converts the oxytocinyl peptides showed a fairly sharp pH dependence with an optimum of 5.5-6, was activated by cobalt ion, and was inhibited by cupric ion, EDTA, and a thiol protease inhibitor, p-chloromercuribenzoate. The amidating activity which converts oxytocinyl-G to oxytocin was competed by degradation due to proteases and/or peptidases present in lysate of Percoll gradient-derived granules. Oxytocinyl-GKR was shown by analytical affinity chromatography to bind noncovalently to neurophysin with an affinity close to that of mature oxytocin. This binding activity and the observation of carboxypeptidase B activity in the presence of large concentrations of neurophysin are consistent with the view that the exoproteolytic processing and amidation steps which occur after initial endoproteolysis of pro-oxytocin/neurophysin likely take place on oxytocin intermediate peptides which are bound in noncovalent complexes with the neurophysin domain from the precursor.
D000818:Animals; D043424:Carboxypeptidase B; D002268:Carboxypeptidases / Q000378:metabolism*; D002417:Cattle; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003594:Cytoplasmic Granules / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007700:Kinetics; D009481:Neurophysins / Q000378:metabolism; D009842:Oligopeptides / Q000138:chemical synthesis; D010121:Oxytocin / Q000031:analogs & derivatives* / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D010904:Pituitary Gland, Posterior / Q000201:enzymology*; D011498:Protein Precursors / Q000138:chemical synthesis / Q000378:metabolism*
D007202:Indicators and Reagents; D009481:Neurophysins; D009842:Oligopeptides; D011498:Protein Precursors; D010121:Oxytocin; C046278:oxytocin, Gly-Lys-Arg-; D002268:Carboxypeptidases; D043424:Carboxypeptidase B