Studies on the chemical modification of the tyrosine residue in bovine neurophysin-II

Abstract

1. Bovine neurophysin-II contains 1mol of tyrosine residue/10000g of protein. This residue could be readily nitrated with tetranitromethane. On hydrolysis and amino acid analysis 1mol of 3-nitrotyrosine was found/10000g of protein. Starchgel electrophoresis at pH8.5 showed that nitration had converted the native protein into a single, more acidic species. The increase in acidity was consistent with the observed fall in pK of the tyrosine hydroxyl from 9.2 in native neurophysin to 7.3 in the nitrated protein. Further, the absence of any intermediate species, even under conditions of minimum substitution, confirmed that the molecular weight of the monomer is 10000. 2. O-Acetylation of the tyrosine residue was carried out with N-acetylimidazole, in conjunction with the reversible blocking of amino groups by citraconylation. The degree of O-acetylation, determined spectroscopically, was 0.9mol of O-acetyltyrosine/10000g of protein. 3. The hormone-binding ability of modified protein was tested by equilibrium dialysis and was found to be unchanged by either nitration or O-acetylation of the tyrosine residue. 4. Interaction of neurophysin-II and [8-arginine]-vasopressin gave rise to a characteristic difference spectrum with a peak at 286.8nm and shoulder at 279.6nm. Part of this hyperchromicity is thought to result from entry of the tyrosine residue at position 2 in the hormone into the hydrophobic environment of the binding site. With nitrated neurophysin-II a second peak appeared at 436nm, showing that the tyrosine of the protein is also perturbed. The very large red shift (84nm) in this region suggests that the 3-nitrotyrosyl residue not only enters a more hydrophobic environment on protein-hormone interaction, but is caused to ionize more fully by the approach of some positively charged group.

Crystal structure of a bovine neurophysin II dipeptide complex at 2.8 A determined from the single-wavelength anomalous scattering signal of an incorporated iodine atom.

“…The crystal structure also confirms the disulfide assignments of Burman et al (7) and is consistent with recent conclusions from solution data as to the relationship of specific protein residues to the principal hormone-binding site. The relationship ofTyr49 to the binding site has been particularly studied because of its marked perturbation by binding (8,38). In the crystal, the suggested proximity of this residue to the binding site (1,39,40) is confirmed, with the backbone atoms of Tyr49 approaching bound peptide at a distance slightly less than 6 A.…”

The Neurohypophysial Hormone‐Binding Proteins: Complete Amino‐Acid Sequence of Ovine and Bovine MSEL‐Neurophysins

“…A role of the single invariant tyrosine, located in position 49, in the binding of the hormones, has been suggested from spectrometric and nuclear magnetic resonance studies [5,37]. On the other hand, because leucine-42 seems to be particularly accessible to chymotrypsin and arginine-43 to trypsin, which split respectively the native protein at the level of these residues, it has been deduced that the central invariant sequence 39-49 is located on the surface of the neurophysins and is likely to be involved in the binding function [38].…”

The Neurophysins

“…This has been demonstrated by studies of the effect of binding on a neurophysin derivative in which the tyrosine is mononitrated (116,121) and by NMR studies, and is discussed in detail in Section V1I.F.…”