No abstract

Thermodynamic binding constants for the interactions of mononitrated neurophysins with oxytocin, vasopressin, and peptide analogues of the hormones were determined by using a spectrophotometric titration technique. The data were fit to a binding model which included all known interactions in these systems. From an examination of the free energies for the binding reaction, we concluded that residues 1-3 contribute 84% of the binding energy for formation of the neurophysin dimer mono complex and 79% for the formation of the bis complex. Rate constants for complex formation and dissociation with native bovine neurophysin were determined by using temperature-jump relaxation. The association rate constants for neurophysin dimer binding to oxytocin, vasopressin, and the peptide analogues were all in the range of 1.3 X 10(6) M-1 s-1 for mono complexation and 1.5 X 10(6) M-1 s-1 for bis complexation. Thus, formation rate constants are identical for both mono and bis complexation, and no significant differences exist between formation constants for hormones and peptides. On the other hand, a clear distinction in dissociation rate constants is apparent when one compares the hormones (kr = 4 to 16 s-1) with the peptide analogues (kr = 54 to 182 s-1). There is rougly a tenfold increase in overall dissociation rate constant when one compares the peptides to the hormones. From these data, we conclude that the rate-determining step in the association reaction involves the first two or three residues on the hormone. After the initial binding takes place, only with intact hormone, i.e., oxytocin or vasopressin, can additional bonding interactions in the complex take place. These additional interactions are reflected in the slower off-rate of the hormone complexes relative to the peptide complexes.

We examined the role of vasopressin (VP) in brain-adrenal function by comparing the plasma corticosterone concentrations of rats genetically deficient in VP [Brattleboro rats, homozygous for diabetes insipidus (Dl-homo)] with those of control animals. Rats were subjected to stressful situations and exogenous ACTH, and their adrenal sensitivity to ACTH was assessed in vitro. Some Dl-homo rats were treated chronically with VP and their plasma corticosterone concentrations were examined during stress. We confirmed that Dl-homo rats have lower than normal plasma corticosterone concentrations during stress and this was at least partially due to adrenal glands relatively insensitive to ACTH.

Gel filtration of an extract of rat median eminence tissue in 0.1 N HCl on Sephadex G-25 separated two peaks, both with feeble ACTH-releasing activity. Full activity of the extract was regained when both peaks were recombined. This observation suggests that the ACTH-releasing hormone of the hypothalamus requires a co-factor for activity.