Abstract
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.
Highlights
Comparisoonf the properties and steps which occurafter initial endoproteolysisof pro- distribution of specific processing enzymes in multiple sites oxytocin/neurophysin likelytake place on oxytocin in- of synthesis of a given set of peptides would allow an improved termediate peptides which are bound in noncovalent understanding of the relationship between molecular events complexes with the neurophysin domain from the preo-ccurring in these sites
The neurohypophysial peptide hormones vasopressin and peptides by enzymes in theneurosecretory granules of bovine oxytocin are synthesized in the hypothalamasupsarts of large posteriorpituitaries.We have detectedthe presence of a precursors, which are post-translationally converted to non- carboxypeptidase activity specific for theCOOH-terminal covalent complexes of the mature hormones and neurophysinbsasic residuesof the oxytocin-containingpeptides oxytocinylduring axonal transport to the posterior pituitary[1,2,3]
E have identified and isolateadcarboxypeptidase frombovine posterior pituitary which is specificfor COOH-terminal basic residues
Summary
Preparation of Neurosecretory Granules-Bovine posterior pituiD taries were obtained from a local abattoir (Trueth and Sons, Catonsville, MD) and transferred to the laboratory on ice. Arose in 0.4 M ammonium acetate, pH 5.7, and to be eluted Reverse-phaseHPLC assay of these conversion reactions with 0.2 M acetic acid This behavior is similar to that foundutilized a linear gradient of 100% TEAP at zero time to 60%. Conversion of Oxytocinyl Peptides OT-GKR and OTK-GK At 96 h, the proportion of product in the form of OT-G was with NeurosecretoryGranule Lysate-The degradation of OT- 30%in the absencoef neurophysin, about15% in itspresence.
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