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The molecular basis of autosomal dominant neurohypophyseal diabetes insipidus, a hereditary deficiency of vasopressin, was determined by nucleotide sequence analysis of the arginine vasopressin-neurophysin-II gene. A C-->T mutation at nucleotide 1761 was detected in one allele of this gene in each affected individual in three generations of one family. This mutant gene encodes a normal arginine vasopressin peptide, but predicts a substitution of leucine for proline at amino acid 24 of neurophysin-II, the arginine vasopressin carrier protein. This mutation was not detected 50 control individuals, thus demonstrating that it is not a common silent genetic polymorphism. The disease arose in the second generation of the studied family, and the chromosome 20 carrying this new mutation was identified by polymorphic CA microsatellite haplotype analysis. The first affected individual inherited this chromosome segment from her mother, who had neither the disease nor this mutation in her somatic cell DNA. Third generation individuals who subsequently inherited this mutation were affected. These data demonstrate that this amino acid substitution in neurophysin-II causes the disease. Two possibilities to explain the mechanism by which clinical deficiency of arginine vasopressin develops even in the presence of one normal arginine vasopressin-neurophysin-II allele are discussed.

The two types of neurophysins known in vertebrate species, namely MSEL-neurophysin (vasopressin-like hormone-associated neurophysin) and VLDV-neurophysin (oxytocin-like hormone-associated neurophysin) have been purified from the pollack (Pollachius virens) pituitary through a combination of molecular sieving and high-pressure liquid chromatography (HPLC). Homogeneity has been checked by gel electrophoresis and return in HPLC. The apparent molecular masses measured by SDS-electrophoresis are near 12 kDa, significantly higher than those found for their mammalian homologues (10 kDa). The two types of neurophysins have been recognized through their N-terminal amino acid sequences. The primary structure of MSEL-neurophysin has been partially determined using automated Edman degradation applied on native and reduced-alkylated protein, as well as peptides derived by trypsin or staphylococcal proteinase hydrolyses. Comparison of pollack MSEL-neurophysin with ox, goose and frog counterparts reveals that particular positions in the polypeptide chain are subjected to substitutions and that the numbers of substitutions do not seem closely related to the paleontological times of divergence between the different vertebrate classes.

Neurophysin is an allosteric protein in which peptide binding and self-association are positively linked. Reaction of neurophysin with succinic anhydride led to a large decrease in peptide affinity assignable to succinylation of a serine or threonine hydroxyl group. To identify the residue involved, acetimidated protein was reacted with [14C]succinic anhydride and the active and inactive components were separated by affinity chromatography. Performic acid oxidation and tryptic and Asp-N mapping of the two components, followed by automated Edman degradation, allowed identification of the critical residue as Ser-56. This residue is not a direct participant in peptide binding and is distant from the subunit interface of the dimer, but it is immediately adjacent to the site of one of the known mutations associated with familial diabetes insipidus. Examination in solution of the peptide affinity of neurophysin succinylated at Ser-56 indicated a binding affinity approximately 1/20th that of the native protein or of protein succinylated at other residues, and a loss of the normal dependence of binding affinity on protein concentration. Under the same buffer conditions, loss of the concentration dependence of binding, in addition to the previously demonstrated loss of binding affinity, also accompanied excision of residues 1-8, an effect attributed to the loss of binding site residue Arg-8. However, in contrast to the effects of succinylation on native neurophysin, only minor effects of succinylation on the binding affinity of the des-1-8 protein were observed.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of neurophysin dimerization on Tyr-49, a residue adjacent to the hormone-binding site, was investigated by proton NMR in order to analyze the basis of the dimerization-induced increase in neurophysin hormone affinity. Dimerization-induced changes in Tyr-49 resonances, in two unliganded bovine neurophysins, suggested that Tyr-49 perturbation is an intrinsic consequence of dimerization, although Tyr-49 is distant from the monomer-monomer interface in the crystalline liganded state. To determine whether this perturbation reflects a conformational difference between liganded and unliganded states that places Tyr-49 at the interface in the unliganded state, or a dimerization-induced change in secondary (2 degrees) or tertiary (3 degrees) structure, the more general structural consequences of dimerization were further analyzed. No change in 2 degrees structure upon dimerization was demonstrable by CD. On the other hand, a general similarity of regions involved in dimerization in unliganded and liganded states was indicated by NMR evidence of participation of His-80 and Phe-35 in dimerization in the unliganded state; both residues are at the interface in the crystal structure and distant from Tyr-49. Consistent with a lack of direct participation of Tyr-49 at the monomer-monomer interface, dimerization induced at least two distinct slowly exchanging environmental states for the 3.5 ring protons of Tyr-49 without significantly increased dipolar broadening relative to the monomer. Two environments were also found in the dimer of des-1-8 neurophysin-I for the methyl protons of Thr-9, another residue distant from the monomer-monomer interface and close to the binding site in the liganded state.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding and spectroscopic properties of ostrich neurophysins were examined with emphasis on the behavior of Tyr-35, a residue that provides a potential probe of the monomer-monomer interface and of allosteric interrelationships between this region and the binding site. Mesotocin-associated ostrich neurophysin was found to bind oxytocin and related peptides with affinities comparable to the mammalian proteins, but induced a significantly different optical activity in bound peptides than the mammalian proteins. Gel-filtration studies indicated higher dimerization constants for the ostrich neurophysins than for the bovine neurophysins. Consistent with this, Tyr-35 was found to be largely buried, as monitored by tyrosine titration and lack of reactivity towards tetranitromethane under non-denaturing conditions. Reaction of Tyr-35 of the mesotocin-associated protein with tetranitromethane under denaturing conditions, followed by refolding, allowed isolation of an active product with an altered interface region as partially evidenced by its titration properties and consistent with its markedly altered CD spectrum. Comparison of the CD spectra of the modified and native proteins and analysis of pH effects indicated the contribution of Tyr-35 to an unusual 237 nm band in the mesotocin-associated protein. Small shifts in the 350 nm CD band of nitrated Tyr-35 on binding peptide and apparent effects of nitration on the induced optical activity in bound peptide provided evidence of at least weak structural communication between Tyr-35 and the binding site. However, no significant effect of nitration on binding affinity was observed, suggesting that, in the mesotocin-associated protein, the region around residue 35 is not a stringent modulator of the thermodynamic behavior of the binding site.

A bovine neurophysin II S-methyl-Cys-Tyr-Phe-NH2 complex has been crystallized using ammonium sulfate as the precipitating agent. The crystals are orthorhombic, the space group is either I222 or I2(1)2(1)2(1) with a = 124.9 A, b = 69.6 A and c = 151.5 A. The crystals diffract to at least 3.0 A resolution. Based on one neurophysin tetramer per asymmetric unit, the Matthews coefficient is calculated to be 3.92 with a solvent content of 69%.

Single crystals of a bovine neurophysin II-oxytocin complex have been obtained using (NH4)2SO4 as the precipitating agent. The crystals diffract to at least 2.7 A resolution, belong to Laue group 4/mmm and exhibit systematic absences consistent with either space group P4(1)2(1)2 or P4(3)2(1)2. The cell dimensions are a = b = 69.07 A and c = 113.26 A. The crystals contain one neurophysin-oxytocin dimer per asymmetric unit. Based on a Vm of 2.9 A3/Da, the solvent content is calculated to be 58%. Chromatographic analysis of the dissolved crystals suggests the presence of three oxytocin molecules per neurophysin dimer.

The mechanism of peptide-enhanced neurophysin self-association was investigated to address questions raised by the crystal structure of a neurophysin-dipeptide complex. The dependence on protein concentration of the binding of a broad range of peptides to the principal hormone-binding site confirmed that occupancy of this site alone, and not a site that bridges the monomer-monomer interface, is the trigger for enhanced dimerization. For the binding of most peptides to the principal hormone-binding site on bovine neurophysin I, the affinity of each dimer site was at least 10 times that of monomer under the conditions used. No interactions between the two sites of the dimer were evident. Fluorescence polarization studies of pressure-induced dimer dissociation indicated that the volume change for this reaction was almost 4 times greater in the liganded than in the unliganded state, pointing to a significant alteration of the monomer-monomer interface upon peptide binding. Novel conformational changes in the vicinity of the single neurophysin tyrosine, Tyr-49, induced by pressures lower than required for subunit dissociation, were also observed. The bovine neurophysin I dimer therefore appears to represent an allosteric system in which there is thermodynamic and functional communication between each binding site and the monomer-monomer interface, but no communication across the interface to the binding site of the other subunit. A model for the peptide-enhanced dimerization is proposed in which intersubunit contacts between monomers reduce the large unfavorable free energy associated with binding-induced intrasubunit conformational change. Structural origins of the lack of communication across the interface are suggested on the basis of the low volume change associated with dimerization in the unliganded state and monomer-monomer contacts in the crystal structure. Potential roles for the peptide alpha-amino group and position 2 phenyl ring in triggering conformational change are discussed.

The crystal structure of a dipeptide complex of bovine neurophysin II has been solved at 2.8 A resolution solely by using single-wavelength anomalous scattering data from a single iodinated derivative. The asymmetric unit is an elongated tetramer of dimensions 110 x 40 x 30 A, composed of two dimers related by pseudo twofold symmetry. Each monomer consists of two homologous layers, each with four antiparallel beta-strands. The two regions are connected by a helix followed by a long loop. Monomer-monomer contacts involve antiparallel beta-sheet interactions, which form a dimer with two layers of eight beta-strands. One peptide per monomer occupies the principal hormone-binding pocket formed by part of the amino-terminal region and parts of the connecting helix and loop, with binding to protein consistent with conclusions drawn from solution studies. Dimer-dimer contacts involve the Tyr49 region adjacent to this site. A fifth dipeptide, of unknown biological significance, helps to stabilize one of the monomer-monomer interfaces and the tetramer-tetramer network in the crystal.

Goose VLDV-neurophysin (mesotocin-associated neurophysin) has been purified from posterior pituitary glands through molecular sieving on Sephadex G-75 and high-pressure reverse-phase liquid chromatography on Nucleosil C-18 columns. Despite apparent molecular mass of unreduced VLDV-neurophysin measured by polyacrylamide gel electrophoresis with sodium dodecylsulfate appeared near 17 kDa, this value fell to 11 kDa after reduction with mercaptoethanol, suggesting the existence of a homodimer. Complete amino acid sequence (93 residues) of goose VLDV-neurophysin has been determined. N- and C-terminal sequences of the protein have been established by Edman degradation (microsequencing) and use of carboxypeptidase Y, respectively. Peptides derived from oxidized or carboxamidomethylated neurophysin by trypsin or staphylococcal proteinase hydrolyses have been isolated by high-pressure liquid chromatography and microsequenced, allowing determination of the complete sequence. Comparison within the vertebrate VLDV-neurophysin lineage, namely goose VLDV-neurophysin to mammalian VLDV-neurophysins and to deduced toad VLDV-neurophysin, reveals a residue insertion between positions 66 and 67 in the nonmammalian VLDV-neurophysins. When goose MSEL-neurophysin (vasotocin-associated neurophysin) and goose VLDV-neurophysin are compared to their bovine counterparts, identical substitutions are found in positions 17 (Asn in both goose neurophysins instead of Gly in both ox neurophysins), 18 (Arg instead of Lys), 35 (Tyr instead of Phe), and 41 (Thr instead of Ala). Identity of the sequences 10-74 in both ox neurophysins has been explained by partial gene conversion between oxytocin and vasopressin genes, and identical substitutions in both goose neurophysins might reveal a similar gene conversion between mesotocin and vasopressin genes in birds.

Quantitative expressions have been derived for bivalent equilibria with immobilized ligand systems and for the equilibria for an immobilized protein whose self-association is modified by binding with a soluble ligand, as analyzed by affinity chromatography. These general expressions have been applied in a reexamination of multivalency in the affinity chromatography of antibodies, as reported by Eilat and Chaiken (Biochemistry 18 (1979) 790) and also to studies of neurophysin-peptide hormone interactions using glass matrices reported by Swaisgood and Chaiken (Biochemistry 25 (1986) 4148).

Vasotocin-associated neurophysin (MSEL-neurophysin) has been purified from goose neurohypophysis through molecular sieving and high-pressure reverse-phase liquid chromatography (HPLC). The protein has a molecular mass (measured by SDS-polyacrylamide gel electrophoresis) of 17 kDa in contrast to 10 kDa found for the mammalian MSEL-neurophysins. Complete amino acid sequence (131 residues) has been determined mainly through tryptic or staphylococcal proteinase peptides derived from carboxyamidomethylated neurophysin, isolated by HPLC and microsequenced. N- and C-terminal sequences have been established by Edman degradation or action of carboxypeptidase Y, respectively, applied on the native protein. Goose MSEL-neurophysin is homologous to the two-domain "big" MSEL-neurophysin previously identified in the frog. It appears that in non-mammalian tetrapods, namely birds and amphibians, the proteolytic processing of the pro-vasotocin involves only one cleavage, releasing the hormone moiety and a "big" neurophysin with two domains homologous to mammalian MSEL-neurophysin and copeptin, respectively. Comparison of the avian protein with its mammalian and amphibian counterparts reveals that the first half of the polypeptide chain is evolutionarily much less variable than the second and that the goose protein resembles the frog protein much more than the mammalian one.

A distinctive property of oligomeric and self-associating proteins is the high specificity of the subunit recognition process. Protein subunits immobilized covalently on a solid matrix maintain this characteristic and are able to bind soluble subunits of the same or a closely related protein under conditions that allow the establishment of a finite association/dissociation equilibrium. The basic theory for studying the immobilized-soluble subunit interaction is presented together with the methodology for a proper protein immobilization. Specific examples are discussed to illustrate on the one hand benefits and caveats of using immobilized protein subunits to measure interaction constants, and on the other preparative applications of subunit affinity columns.

Unlabelled: Xenopus laevis neurophysins have been purified from neurointermediate pituitaries through high-pressure reverse-phase liquid chromatography and their N-terminal amino acid sequences have been determined by microsequencing. Two types of neurophysins, corresponding to mammalian MSEL- and VLDV-neurophysins, have been distinguished. A strong homology exists between neurophysins of Xenopus (Pipidae), frog (Ranidae) and toad (Bufonidae). Xenopus MSEL-neurophysin, as frog MSEL-neurophysin, has a high molecular mass suggesting that the C-terminal domain of the vasotocin precursor is not processed in contrast to the two-step processing observed for mammalian vasopressin precursor., Abbreviations: Mammalian neurophysins are termed MSEL- and VLDV-neurophysins according to the nature of residues in positions 2, 3, 6 and 7 (one-letter symbols for amino acids).

The methyl-acceptor activities of bovine neurophysins I and II for the enzyme protein carboxymethylase (EC 2.1.1.24) were found to be similar and as high as for other previously identified, biologically active protein substrates. Effects on the rate of methylation of these neurophysins were investigated with the posterior pituitary hormone ligands, oxytocin and vasopressin, and the hormone-related tripeptide ligand, methionyl-tyrosyl-phenylalaninamide. An increase in the rate of neurophysin II methylation was observed with both oxytocin and tripeptide. This ligand-induced response did not occur with either native neurophysin I or disulfide-scrambled neurophysin II.

Purification of 125I-labelled lysine-vasopressin has been achieved by affinity chromatography on a Sepharose 4 B conjugate of porcine neurophysins. This affinity absorbent did not retain halogenated hormone, while native lysine-vasopressin was bound on the solid support. The specific activity of purified iodinated lysine-vasopressin was 1700-1800 Ci/g, corresponding to one iodine atom per mole. By comparison with an unpurified tracer, a five times increase in the minimum detectable dose was obtained in the vasopressin radioimmunoassay.