Hormonal Interactions at the Molecular Level

Abstract

Bovine neurophysins I and I1 have been obtained in a highly purified state using isoelectric focusing. The interactions of oxytocin and [8-lysine]vasopressin with these proteins have been investigated by thin-film equilibrium dialysis using highly radioactive hormones retaining their full biological activities. I n this report, we present the results of binding studies carried out using the equilibrium dialysis technique with highly purified neurophysins obtained by isoelectric focusing of crude protein material. The thermodynamic parameters and the stoichiometry of the reaction between the hormones and the proteins have been established. These findings are discussed in terms of the molecular mechanisms by which the neurophysins bind to the hormonal peptides.

125I‐d(CH2)5[Tyr(Me)2,Tyr(NH2)9]AVP: Iodination and binding characteristics of a vasopressin receptor ligand

“…Specific radioactivity was 23.3 Ci/mmoJ. The labelled peptides were purified by HPLC and affinity-chromatography using a neurophysin-Sepharose column [8]. AVP was purchased from Bachem (Bubendorf, Switzerland).…”

13C nuclear magnetic resonance studies of the interactions of bovine neurophysins with (1‐hemi‐[1‐13C]cystine)oxytocin and (1‐hemi‐[1‐13C]cystine, 8‐arginine)vasopressin

“…The different value of the pK, of the free hormone as compared to the pK, which affects binding, may be due to the different conditions under which these pK, values were determined. Also the actual pK, which influences the binding constant is best determined as described by Dixon [17] i.e., plotting log K versus pH, rather than K versus pH [5].…”

“…Several studies [l-5] indicate that the o-amino group of the hormones is intimately involved in the interaction with the proteins. Binding studies of neurohypophyseal hormone-neurophysin interaction have indicated that the binding constant is pH-dependent [2,5-71 and a plot of binding constants versus pH [5] yields a bell shaped curve, with pK, values of about 4.5 and 7. The pK, of the a-amino group of the free hormones is about 6.3 [4] and the pK, of 7 observed in the presence of neurophysin was suggested to represent the pK, of this group in the protein--hormone complex.…”

Neurophysins, rather than Receptors, are Involved in [3H]Oxytocin and [3H]Vasopressin Binding Detected by Autoradiography in the Hypothalamo‐Neurohypophyseal System

“…In a previous study, we detected [3H]OT binding in the magnocellular nuclei of Long Evans rats (1 1) but not of Wistar rats, which contain less neurohypophyseal hormones and secretory vesicles than the Long Evans strain (9). However, as already pointed out ( I 9, 20), the interaction characteristics of the neurohypophyseal peptides with neurophysins, such as a K, above the micromolar range and optimal binding around pH 5.5 (26,27), should not permit tritiated peptides to bind to the neurophysins under the conditions used in autoradiographic studies (tritiated peptide concentrations in the nanomolar range, pH 7.4). Moreover, on the basis of displacement studies, Dashwood and Robinson concluded that [3H]VP detected by autoradiography in the posterior pituitary was bound to specific VP receptors associated with normal synthesis, packaging and/or transport of VP granules to the neural lobe (22).…”

“…Various experiments were performed to discriminate between binding of neurohypophyseal hormones to specific receptor sites and/or on neurophysins: 1) saturation experiments using increasing doses of [3H]OT and [3H]VP; 2) competition experiments with selective ligands; 3) binding of an iodinated OT antagonist with no affinity for neurophysins (30); 4) incubation in the absence of MgCl, which impedes OT and VP binding to receptors but does not affect their interaction with neurophysins (26,27). OT and VP binding were also studied in the neural lobes ofdehydrated rats in which the neurosecretory vesicles are dramatically decreased (31).…”