The preparation and properties of porcine neurophysin and the influence of calcium on the hormone‐neurophysin complex

Abstract

SUMMARY1. The preparation of a hormone-binding protein fraction (neurophysin) from porcine neurohypophyses is described.2. It was shown by gel-filtration that the protein (which sedimented as a single component on analytical ultracentrifugation) forms complexes with oxytocin and lysine vasopressin. The maximum capacity of porcine neurophysin to bind oxytocin and lysine vasopressin was estimated from the results of dialysis experiments as 232 u. oxytocin and 48 u. lysine vasopressin/mg protein.3. Binding of oxytocin and lysine vasopressin by neurophysin was completely inhibited in the presence of calcium in concentrations greater than 106 M but is unaffected at less than 10-7 m-Ca. The concentration of calcium required to inhibit binding did not appear to be dependent on hormone or protein concentrations.4. No evidence was obtained that calcium was bound by neurophysin or by the peptide hormones either alone or in combination.5. These results are discussed in the light of the hypothesis that release of the hormone from the neurohypophysis is mediated by calcium.

Polymerizing equilibria in neurophysin

“…high molecular weight) being favoured. However, in experiments in which Ca2+ (which does not bind to neurophysin at pH 5-8, Ginsburg et al 1966) is gradually removed from the protein during its passage through the column, the trailing edge elution profile was indicative of polymerization. It would appear, therefore, that the effects of calcium on the protein do not survive removal of the cation.…”

Hormonal Interactions at the Molecular Level

“…Some authors [30,31] have suggested that Ca2+ ions would inhibit the binding of polypeptide hormones to neurophysins possibly playing a role in the release of those peptides from their complexes with the proteins. Optical rotatory dispersion [32] and titration [6] measurements allowed Breslow et al to show that Ca2+ had apparently no measurable effect on the formation of neurophysin 11 -oxytocin complex.…”

The Neurophysins

“…(Using an average molecular weight of 10,000 per monomer for bovine neurophysin, this reduces to 2.8 moles of oxytocin and 1.6 moles of arginine vasopressin per monomer.) Similar studies on binding to a porcine neurophysin (64,80) suggested that 14 moles of oxytocin and 4 moles of lysine vasopressin were bound per 25,000 g. Although, as will be seen, these figures are undoubtedly too high, they suggest that neurophysin has a greater capacity for binding oxytocin than it does for binding vasopressin. Pickering (IS) has interpreted his binding studies with cod neurophysins also to indicate a greater capacity for the binding of oxytocin than of vasopressin.…”

“…Both mechanisms utilize the observation by Douglas and Poisner (76,77) that release is accompanied by, and dependent on, an increase in intraneuronal Ca" concentration. The hormone-dissociation hypothesis was elaborated by Thorn and Smith (78,79) and Ginsburg et al (64,80,81) and is based on reports (78-80) that Ca2+ ion, at concentrations comparable to those attained within the neuron on stimulation, weakened the affinity of neurophysin for oxytocin and vasopressin. According to this hypothesis, the intraneuronal entry of Ca" that attends nerve stimulation dissociates the hormones from neurophysin so that they diffuse from within the neurosecretory granules into the extragranular space and out of the neuron.…”