Interdependence of neurophysin self-association and neuropeptide hormone binding as expressed by quantitative affinity chromatography

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Abstract
The reciprocal modulation of neurophysin self-association and noncovalent peptide-protein interaction between neurophysin and the hormones oxytocin and vasopressin was assessed by quantitative affinity chromatography. Competitive elutions of radiolabeled bovine neurophysin II (NPII) from the affinity matrices Met-Tyr-Phe-.omega.-(aminohexyl) [and (aminobutyl)-] agarose were performed with increasing concentrations of either of the soluble ligands oxytocin or lysine-vasopressin. Also, the dependence of NPII retardation by the same adsorbents on the concentration of applied protein was investigated in the absence of soluble ligand. The affinity constant of NPII for the immoblized peptide increased markedly with increasing amounts of applied protein and with the addition of small amounts of soluble ligand, the latter being more pronounced at higher protein concentrations. The affinity constant of the protein for the soluble ligand showed a smaller increase. The variation of 1/(V - V0) (where V = the NPII elution volume and V0 = the elution volume of noninteracting control protein) with soluble ligand concentration was linear except near [ligand] = 0. The quantitative affinity chromatographic results on the tripeptidyl affinity columns are consistent with the view tha NPII exists in a monomer .dblarw. dimer equilibrium, with the dimer exhibiting a stronger interaction with both neuropeptide and tripeptide analogs. The self-associated protein dimer itself exhibits cooperativity, that is, stronger binding of the immobilized ligand at one site when a second site is occupied with a molecule of the soluble ligand than when no soluble ligand is bound. The deduction from the above of ligand-induced dimerization is evident also in the increased retardation of NPII on neurophysin-Sepharose when the eluting buffer contains soluble peptide hormone.