J. Biol. Chem., Vol. 263, Issue 2, 769-775, Jan, 1988
Effect of neurophysin on enzymatic maturation of oxytocin from its precursor
S Ando, AS Murthy, BA Eipper and IM Chaiken
Molecular, Cellular, and Nutritional Endocrinology Branch, National Institutes of Health, Bethesda, Maryland 20892.
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.
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