J. Biol. Chem., Vol. 260, Issue 18, 10118-10124, Aug, 1985
Pituitary enzyme conversion of putative synthetic oxytocin precursor intermediates
T Kanmera and IM Chaiken
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.
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