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

Abstract

The reciprocal modulation of neurophysin self-association and noncovalent peptide--protein interaction between neurophysin and the hormones oxytocin and vasopressin has been assessed by quantitative affinity chromatography. Competitive elutions of radiolabeled bovine neurophysin II (NPII) from the affinity matrices Met-Tyr-Phe-omega-(amino-hexyl)- [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 immobilized 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 l/(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 that NPII exists in a monomer in equilibrium dimer equilibrium, with the dimer exhibiting a stronger interaction with both neuropeptide and tripeptide analogues. The data also indicate that 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.

Interaction between immobilized and soluble protein subunits. Analysis and applications

“…The close agreement with literature values for the soluble protein (Whittaker and Allewell, 1984) shows that immobilization on the high performance affinity supports described above does not alter the self-association properties of neurophysin, at least statistically. In contrast, with Sepharose-bound neurophysin, a dimerization constant that is roughly 10-fold higher than in solution was obtained (Angal and Chaiken, 1982;Chaiken et a/., 1983). This result is unexpected since any disruption of the recognition surfaces that may occur on immobilization can be foreseen to decrease the association capacity of the insoluble material.…”

“…Bovine neurophysin I1 has been immobilized on supports of a different nature, namely Sepharose 4B (Angal and Chaiken, 1982;Chaiken et al, 1983) and more recently on several pressure-resistant affinity supports, such as nonporous glass (NPG) beads , ACCELL (polyacrylamide-coated silica, from Waters) and highly cross linked (HXL) agarose (from LKB) (Fassina et al, 1986;Fassina and Chaiken, 1988), that are suitable for use with high performance liquid chromatographic instrumentation. To this end the immobilized protein was packed in an HPLC column and used as an analytical tool in an HPLC system.…”

Cooperative interactions in neurophysin‐neuropeptide hormonecomplexes

“…The preparation of affinity matrices used for this study has been described elsewhere (23). Bed volumes and immobilized polypeptide concentrations for these matrices were 2.5 ml and 0.30pmol peptide/ml of bed volume for Met-Tyr-Phe-AB-agarose and 2 ml and 0.…”

“…Amino acid analysis of the final product indicated that the overall yield of [3-nitro-Tyr 491 BNP-I1 was approximately 30% of the starting NP-11. Native neurophysins and [3-nitro-Tyr 491 BNP-I1 were I-labeled by reacting at amino groups using Bolton-Hunter reagent (Nsuccinimidyl-3(4-hydroxy-5-[ I] iodophenyl) propionate) (23,38). After reaction the iodinated proteins were purified by Sephadex (3-25 chromatography in 0.4 M ammonium acetate, pH5.7, followed by affinity chromatography on Met-Tyr-Phe-AH-agarose (23).…”

“…Native neurophysins and [3-nitro-Tyr 491 BNP-I1 were I-labeled by reacting at amino groups using Bolton-Hunter reagent (Nsuccinimidyl-3(4-hydroxy-5-[ I] iodophenyl) propionate) (23,38). After reaction the iodinated proteins were purified by Sephadex (3-25 chromatography in 0.4 M ammonium acetate, pH5.7, followed by affinity chromatography on Met-Tyr-Phe-AH-agarose (23). Tryptic protein fragments of BNP-I, denoted BNP-I(9-93) and [des [19][20] BNP-I(9-93), were produced by digestion of native BNP-I (I mg/ml) with TPCK-trypsin (13.3% by w e a t ) in 0.1 M ammonium bicarbonate, pH8.6, at 37' for 1.5h (39).…”