Physical and Chemical Properties of the Bovine Neurophysins

• Published in: The Journal of biological chemistry Vol. 246; no. 17; pp. 5179 - 5188
• Main Authors: Breslow, E, Aanning, H L, Abrash, L, Schmir, M
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 10.09.1971
• Subjects: Alanine; Amides; Amino Acids - analysis; Animals; Binding Sites; Calcium; Cattle; Chromatography, Gel; Chromatography, Ion Exchange; Circular Dichroism; Cystine; Dipeptides; Electrophoresis, Disc; Glycine; Histidine; Hydrogen-Ion Concentration; Macromolecular Substances; Methionine; Models, Biological; Molecular Weight; Oxytocin; Peptides; Phenylalanine; Pituitary Gland, Posterior; Potentiometry; Protein Binding; Protein Conformation; Proteins - analysis; Proteins - isolation & purification; Spectrophotometry; Tyrosine; Ultracentrifugation; Ultraviolet Rays; Vasopressins
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)61892-7

The amino acid composition of native bovine neurophysins I and II, isolated by 0.1 n HCl extraction of acetone-desiccated posterior pituitary glands, has been contrasted with that of neurophysins isolated under conditions of lesser acidity. The results indicate that one of the principal fractions isolated under the latter condition represents neurophysin II from which the amino-terminal Ala-Met peptide has been removed by proteolytic digestion. The previously shown ability of this fraction to bind oxytocin and vasopressin indicates that residues 1 and 2 of neurophysin II, which include its single methionine residue, are nonessential to hormone-binding. Molecular weight studies of neurophysins I and II indicate that they are heterogeneous with respect to molecular weight and that neurophysin II is a mixture of monomer and dimer under the conditions studied. Circular dichroism studies indicate that the conformations of neurophysins I and II are similar and suggest almost no α-helical content. H + ion titration studies indicate that all ionizable side chains in neurophysin II, in addition to the single histidine of neurophysin I, are freely available to solvent. Changes in proton equilibria and circular dichroism accompanying neurophysin-hormone interaction have been used to study the interaction of neurophysin with a series of peptides containing sequences related to those at positions 1 to 3 of oxytocin and vasopressin. The results indicate that the principal qualitative features of neurophysin-hormone interaction are preserved in its interaction with peptides containing only the first 3 residues of the hormones. In addition, the following relative binding affinities to neurophysin were observed: l -cystinyl-bis- l -tyrosine amide, l -methionyl- l -tyrosyl- l -phenylalanine amide > S -methyl- l -cysteinyl- l -tyrosyl- l -phenylalanine amide > l -alanyl- l -tyrosyl- l -phenylalanine amide > glycyl- l -tyrosyl- l -phenylalanine amide. These data indicate that, together with the hormone α-NH 2 and its side chains in positions 2 and 3, the side chain attached to the α-carbon at position 1 plays an important role in binding to neurophysin. This finding is tentatively interpreted in terms of a model in which water is excluded from the binding site upon interaction. H + ion titration studies confirm previous conclusions from optical activity studies that high concentrations of calcium ion do not significantly diminish the interaction of the hormones with bovine neurophysin. These results differ from the reported effect of calcium ion on porcine neurophysin-hormone interaction.