Interaction of bovine neurophysin with oxytocin and vasopressin measured by temperature-jump relaxation

Abstract

The interaction between bovine neurophysins I and II and oxytocin and vasopressin was measured using temperature-jump relaxation. A single relaxation time in the 10 to 90 ms range was noted for each solution. This time depended upon the concentration of both neurophysin and hormone and increased with increasing pH. The formation rate constants (+/- SE) for the interaction of neurophysin I dimer with the protonated form of oxytocin and vasopressin at pH 7.4 in 0.1 M KNO3 and 25 degrees C were 2.8 (+/- 0.4) x 10(6) and 2.3 (+/- 0.5) x 10(6) M-1 s-1, respectively; the dissociation rate constants were 11 and 15 s-1, respectively.For neurophysin II dimer, formation rate constants were 6.0 (+/- 0.2) x 10(6) and 2.4 (+/- 0.3) x 10(6) M-1 s-1; dissociation rate constants were 24 and 16 s-1 for oxytocin and vasopressin, respectively. Formation rate constants for the interaction of neurophysin monomer with protinated hormone were approximatley an order of magniment with circular dichorism and pH titration studies which indicate that this interaction occurs between a negatively charged carboxyl group on the neurophysin and a protonated alpha-amino group on the hormone. Our results indicate that oxytocin and vasopressin bind with greater affinity to the neurophysin dimer than the monomer and that the binding of oxytocin and vasopressin in neurophysin dimer than the monomer and that the binding of oxytocin and vasopressin in neurophysin dimer at pH 7.4 and concentrations between 10(-4) and 10(-5) M is nearly identical for each hormone.

Cooperative binding of ocytocin to bovine neurophysin A comparison of two theoretical models

“…The essential features of the process were confirmed [9,12]. The theoretical curves obtained from this model (model l), gave good results for the protein concentration dependence of the average slopes of the binding isotherms.…”

“…(ii) The higher affinity (5-fold) of ocytocin for the neurophysin dimer than for the monomer [9]. (iii) The predominance of dimeric complexes under normal binding conditions [12]. The discrepancies between our dimeric association constants ratio values (K'2[K2 = 4) and those in [10] about the binding of ocytocin to neurophysin II at pH 5.8, or those in [9] (K'2/K2 = 2) about the binding of the dipeptide Phe-Tyr-NH2 to mononitrated neurophysin II at pH 6.2, provide a further illustration of the observation that it may be rather innacurate to consider a restricted protein concentration range when analyzing results in terms of a particular model, especially a polymerizing one.…”

Molecular, Thermodynamic, and Biological Aspects of Recognition and Function in Neurophysin‐Hormone Systems: A Model System for the Analysis of Protein–Peptide Interactions

“…All estimates so far of the relative peptide affinities of unliganded monomer and dimer indicate that the unliganded dinier has a higher affinity for the first bound peptide than does the monomer (3, 80). Moreover, the peptide-association rate constant of the monomer is considerably slower than that of dimer (63,64,145), which is not explained by this model.…”

“…This conclusion does not invalidate the model, however, since the 6 to 7 fold affinity difference between monomer and dimer is energetically small and is potentially explained by local conformational differences. The finding that the peptide association and dissociation rate constants for binding to monomer are both slower than for binding to the dimer (63,64,145) is also most simply explained by this model. Additionally, the assumption that the rate-determining step in dimerization and in peptide binding to the monomer is the same conformational change (Fig.…”

Properties of CRF from Normal and Brattleboro Rat Median Eminence

“…Bovine neurophysins I and II were purified from freeze-dried posterior pituitaries (Pel-Freez) as previously described [9). 3 g of cyanogen-bromide-activated Sepharose 4B (Pharmacia) was reacted with 30 mg of bovine neuro physins I and II.…”