Presence of immunoreactive neurophysins of a higher molecular weight in addition to the 10.000 form in the ovine pineal gland

Using an aqueous extraction followed by ultrafiltration through Amicon Diaflo membranes, two ovine pineal fractions were obtained, which contain immunoreactive neurophysin. The presence of neurophysin was monitored by radioimmunoassay, employing an antiserum raised against pituitary bovine neurophysin and selected because it reacts with neurophysins of many other mammals. From 50 g of wet ovine pineal glands 552 micrograms of immunoreactive neurophysins were obtained. About 5% of these immunoreactive neurophysins are eluted from three different Sephadex columns with an elution volume corresponding to Mr above 10,000 between bovine serum albumin and pituitary neurophysin. The remaining 95% of ovine immunoreactive pineal neurophysin (Mr 10,000) shares immunological and physico-chemical properties with highly purified bovine pituitary neurophysin used as a reference. From the results of gel filtration and affinity chromatography on LVP-Sepharose it was concluded that ovine pineal gland may contain a neurophysin precursor molecule in addition to the neurophysin Mr 10,000.     

Neurophysin in the large luteal cell of the nonpregnant water buffalo (Bubalus bubalis): immunohistochemical localization

Light microscopy immunohistochemistry was used to localize neurophysin in the corpus luteum of the mid-luteal phase of the estrous cycle of the water buffalo (Bubalus bubalis). Corpora lutea weighing 0.39-0.65 g from a recent ovulation showed no staining. Corpora lutea identified with the late luteal phase showed only weak evidence of staining. The neurophysin staining was confined to a specific region of large oval-shaped cells (20-30 microns diameter), which had a very eosinophilic cytoplasm. The intense localization of staining to a distinct area of the cytoplasm was previously only observed in the corpus luteum of the cow. Corpora lutea obtained from all quadrants of pregnancy did not stain. Controls in which the neurophysin antiserum was substituted with serum from an unimmunized rabbit (normal rabbit serum) or neurophysin antiserum preabsorbed with bovine oxytocin-associated neurophysin I also did not stain. These data indicate the neurophysin is present in the mature corpus luteum of the nonpregnant water buffalo as it is in other nonpregnant ruminants, the ewe and cow.     

Immunocytochemical identification of neurophysin-secreting neurons in the hypothalamo-neurohypophysial system of some non-mammalian vertebrates

Antiserum raised against a mammalian neurophysin, porcine neurophysin-II, was used in conjugation with the immunoperoxidase histochemical technique to detect neurophysin in the hypothalamus of the chickens, frog and goldfish. In the chickens, the paraventricular and supraoptic nuceli as well as the internal and external zones of the median eminence stained for neurophysin. Material in the perikarya of the frog and goldfish preoptic nucleus also cross-reacted immunologically against anti-porcine neurophysin-II serum. Serial dilutions of the anti-mammalian neurophysins serum were carried out in order to ascertain at which point the 3-layer immunocytochemical reaction ceased to localize neurophysin. In the chicken, frog and goldfish as well as in the rat, neurosecretory structures became difficult to visualize between 12800 and 25400 fold dilution of antiserum. The results demonstrate that the immunological cross-reactivity previously observed between an anti-mammalian neurophysin serum and the neurophysin isolated from mammals of varying phylogeny also extends to certain non-mammalian vertebrates and is suggestive of a structural homology of neurophysin from different species.     

Ultrastructural immunocytochemical localization of neurophysin and vasopressin in the median eminence and posterior pituitary of the guinea pig

Summary With the use of tissue prepared by freeze-substitution and the unlabelled antibody enzyme technique, neurophysin and vasopressin were localized at the ultrastructural level in the posterior pituitary and median eminence of the guinea pig. In the posterior pituitary neurophysin was found in the large neurosecretory granules (1300–1500 Å) of axons, Herring bodies, and nerve terminals. In some of these axons immunoreactive neurophysin was found outside of granules in the axoplasm. By light microscopy neurophysin was found in both the zona interna and zona externa of the median eminence; this was confirmed by electron microscopy. In the zona interna as in the posterior pituitary, neurophysin was localized both inside and outside the large neurosecretory granules. In the zona externa, immunoreactive deposit was primarily located in granules with a diameter of 900–1100 Å in nerve terminals abutting on the primary portal plexus. The distribution of vasopressin paralleled that of neurophysin except that the hormone was rarely extragranular. These results demonstrate for the first time that both neurophysin and vasopressin are present in granules of axons that are in contact with the hypophysial portal vasculature.The authors wish to thank Dr. Alan Robinson for the gifts of antiserum to bovine neurophysin I and for purified bovine neurophysin I; Dr. Ludwig Sternberger for the peroxidase-anti-peroxidase complex; and Dr. Robert Utiger for antiserum to lysine vasopressinSupported in part by U.S. Public Health Service grant RR-00167 to the Wisconsin Regional Primate Research Center from the National Institutes of Health. Primate Center publication No. 14-017.Recipient of NIH, NINDS Teacher-Investigator Award NS-1108.     

Complete amino acid sequence of bovine neurophysin-I. A major secretory product of the posterior pituitary

Bovine neurophysin-I (bNP-I) is the first neurophysin protein which contains histidine and possesses an acidic COOH-terminal segment for which the complete amino acid sequence is presented: NH2-Ala-Val-Leu-Asp-Leu-Asp-Val-Arg-Thr-Cys-Leu-Pro-Cys-Gly-Pro-Gly-Gly-Lys-Gly-Arg-Cys-Phe-Gly-Pro-Ser-Ile-Cys-Cys-Gly-Asp-Glu-Leu-Gly-Cys-Phe-Val-Gly-Thr-Ala-Glu-Ala-Leu-Arg- Cys-Gln-Glu-Glu-Asn-Tyr-Leu-Pro-Ser-Pro-Cys-Gln-SerGly-Gln-Lys-Pro-Cys-Gly-Ser- Gly-Gly-Arg-Cys-Ala-Ala-Ala-Gly-Ile-Cys-Cys-Ser-Pro-Asp-Gly-Cys-His-Glu-Asp-Pro-Ala-Cys-Asp-Pro-Glu-Ala-Ala-Phe-Ser-Leu-COOH. Determination of the structure was greatly facilitated by new procedures used for the isolation of bNP-I and of its tryptic peptide fragments. bNP-I isolated from freshly frozen bovine posterior pituitaries is composed of 93 residues, but some preparations contain neurophysin protein with NH2- and COOH-terminal truncated sequences. bNP-I differs from bovine neurophysin-II, the second major neurophysin of cow, in 20 residue positions, and several of the differences cannot be accounted for by single nucleotide replacements in the genes coding for these two neurophysin proteins. The results reported in this study support our earlier hypothesis that neurophysin-gene duplication preceded species divergence.     

Immunocytochemical demonstration of separate vasopressin-neurophysin and oxytocin-neurophysin neurons in the human hypothalamus

Summary With the use of immunocytochemistry, it was shown that both the supraoptic and paraventricular hypothalamic nuclei in humans contain at least two different neurophysins. These two human neurophysins are immunologically related to bovine neurophysin I and neurophysin II, respectively. One human neurophysin is associated with vasopressin, the other with oxytocin. Human vasopressin-neurophysin and oxytocin-neurophysin are located separately in two different types of neurons, which correspond respectively to the vasopressinergic and oxytocinergic neurons of both the supraoptic and paraventricular nuclei. The neurophysin of the human vasopressinergic suprachiasmatic neurons appears to be closely related to or identical with neurophysin of the vasopressinergic neurons of the human magnocellular hypothalamic nuclei.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

Expression, folding, and thermodynamic properties of the bovine oxytocin-neurophysin precursor: relationships to the intermolecular oxytocin-neurophysin complex.

Earlier thermodynamic studies of the intermolecular interactions between mature oxytocin and neurophysin, and of the effects of these interactions on neurophysin folding, raised questions about the intramolecular interactions of oxytocin with neurophysin within their common precursor. To address this issue, the disulfide-rich precursor of oxytocin-associated bovine neurophysin was expressed in Escherichia coli and folded in vitro to yield milligram quantities of purified protein; evidence of significant impediments to yield resulting from damage to Cys residues is presented. The inefficiency associated with the refolding of reduced mature neurophysin in the presence of oxytocin was found not to be alleviated in the precursor. Consistent with this, the effects of pH on the spectroscopic properties of the precursor and on the relative stabilities of the precursor and mature neurophysin to guanidine denaturation indicated that noncovalent intramolecular bonding between oxytocin and neurophysin in the precursor had only a small thermodynamic advantage over the corresponding bonding in the intermolecular complex. Loss of the principal interactions between hormone and protein, and of the enhanced stability of the precursor relative to that of the mature unliganded protein, occurred reversibly upon increasing the pH, with a midpoint at pH 10. Correlation of these results with evidence from NMR studies of structural differences between the precursor and the intermolecular complex, which persist beyond the pH 10 transition, suggests that the covalent attachment of the hormone in the precursor necessitates a conformational change in its neurophysin segment and leads to properties of the system that are distinct from those of either the liganded or unliganded mature protein.     

Molecular properties of the oxytocin/bovine neurophysin biosynthetic precursor. Studies using a semisynthetic precursor

An oxytocin/bovine neurophysin I biosynthetic precursor, [N epsilon-diacetimidyl-30,71, des-His106]pro-OT/BNPI, was synthesized from a synthetic oxytocinyl peptide, 1/2Cys-Tyr-Ile-Gln-Asn-1/2Cys-Pro-Leu-Gly-Gly-Lys-Arg, and native neurophysin by chemical semisynthesis. The semisynthetic precursor contains the entire sequence of the biosynthetic precursor deduced from the complementary DNA structure except for omission of the carboxyl-terminal histidine residue. The covalent structure of the semisynthetic product was verified by amino acid analysis and amino-terminal analysis. Analytical affinity chromatography was employed to evaluate noncovalent binding properties of the precursor. The precursor does not bind significantly to immobilized Met-Tyr-Phe, a hormone binding site ligand. In contrast, the acetimidated precursor binds to immobilized bovine neurophysin II, with a 13-fold higher affinity than does acetimidated neurophysin itself. When a hormonal ligand, [Lys8]vasopressin, was added to the elution buffer at the concentration of 0.1 mM so that a major portion of the immobilized BNPII was liganded, the affinity between the immobilized liganded BNPII and the precursor was enhanced 8-fold and approached the affinity for the liganded (bovine neurophysin I-immobilized BNPII) interaction. The data imply that the precursor can self-associate and that this self-association is closely related to that of liganded neurophysin. The tripeptide affinity matrix data argue that, in the precursor, the ligand binding site of the neurophysin domain is occupied intramolecularly by the hormone domain. The data verify the view that both the self-association surface and hormone binding site are established upon precursor folding. A disulfide stability analysis showed the resistance, to disulfide interchange by dithiothreitol, of semisynthetic precursor but not of neurophysin, as judged by protein association and peptide ligand binding activities, respectively. The results argue that the molecular structure of the precursor is established upon precursor folding and before enzymatic processing that produces mature hormone and neurophysin.     

Ultrastructural localisation of oxytocin and neurophysin in the ovine corpus luteum

Summary Polyclonal rabbit antisera raised against oxytocin, bovine neurophysin I and vasopressin were used, together with an immunogold complex, to localise the peptides in ultrathin sections of ovine corpus luteum. The only organelle which consistently showed gold labelling was the secretory granule of the large luteal cell. In non-consecutive sections of the same large luteal cell all the granules showed a similar level of labelling after oxytocin or neurophysin I antisera: however no immunolabelling was detected for vasopressin. Oxytocin and neurophysin seem to be rapidly lost after secretion since exocytosed granule cores showed no labelling above background levels.     

THE DISTRIBUTION OF NEUROPHYSINS AND POSTERIOR PITUITARY HORMONES IN THE PORCINE NEUROHYPOPHYSEAL SYSTEM

Specific, homologous porcine neurophysin I and II radioimmunoassays were established together with specific oxytocin and vasopressin radioimmunoassays. The levels of each of these proteins and peptides were measured in acid extracts of individual paraventricular nuclei, supraoptic nuclei, neurohypophyseal stalks and posterior pituitary lobes of 12 pigs in order to quantitate the neurophysin-hormone relationships in the porcine neurohypophyseal system. Neurophysin III was found to be immunologically identical to neurophysin I. Neurophysin measurements by radioimmunoassay were quantitatively validated by scanning densitometry of polyacrylamide gels stained with 0.5% amido schwarz. In the hypothalamic nuclei vasopressin was in 3–4 M excess of oxytocin but in the neurohypophyseal stalk and posterior pituitary lobe the hormones were equimolar suggesting that the rate of formation of vasopressin differs from that of oxytocin. Neurophysin I immunoreactivity was present in a 3:1 molar ratio with neurophysin II throughout the porcine neurohypophyseal system. In posterior pituitary lobes total neurophysins were equimolar to total hormone concentrations. The specific activity (pmol/mg extracted protein) of oxytocin increased 1800 times, vasopressin 560 times and neurophysins about 360 times from the paraventricular nucleus to the posterior pituitary lobe. In the hypothalamic nuclei relationships between immunoreactive neurophysin I and vasopressin, and between neurophysin II and oxytocin were highly significant. In the posterior pituitary lobe each immunoreactive neurophysin level correlated with both hormone levels. Quantification of densitometric scans of stained polyacrylamide gels from neurophypophyseal extracts and immunoreactivity patterns of neurophysins in eluates of sliced, duplicate gels indicated that neurophysin III decreased distally within the neurohypophyseal tract while neurophysin I increased. The results demonstrated that vasopressin was associated with porcine neurophysin I. However, oxytocin may be associated with both immunoreactive neurophysin I and neurophysin II in the porcine neurohypophyseal system if a 1:1 molar ratio of neurophysin to hormone is to be maintained. Neurophysin III contributed to the stoichiometry of this relationship.     

Highly purified neurophysin proteins: Method of isolation based on their acidic properties

The isolation of highly purified bovine neurophysins I and II from freshly frozen posterior pituitaries is reported. The method can also be used for the isolation of neurophysins from other species, and acetone-desiccated preparations may serve as starting material as well. Crude posterior pituitary extract was obtained as described by Hollenberg and Hope (1967, Biochem. J., 104, 122–127). Basic and neutral proteins were then separated from the acidic neurophysins by cation-exchange chromatography on Cellex-CM (carboxymethyl). Neurophysin I was separated from neurophysin II by anion-exchange chromatography on DEAE-(diethylaminoethyl)-Sephadex with a continuous sodium chloride gradient (0 to 0.4 m). Highly purified bovine neurophysin I was also secured with a stepwise sodium chloride gradient (0.22 m starting gradient followed by a steep gradient from 0.22 to 0.4 m). The current method yields neurophysin proteins in a higher overall yield than previous procedures, as determined by single radial immunodiffusion and concentration-dependent absorption after disc electrophoresis. The method also gives neurophysins of greater purity than standard procedures currently in use. The proteins are characterized by a single, sharp precipitation band on immunodiffusion and immunoelectrophoretic analysis against antiporcine neurophysin antibody, by single bands on analytical gel disc electrophoresis at a running pH of either 8.8, 5.9, or 4.0. Isoelectric focusing on polyacrylamide gel gave an apparent pI value of 4.31 ± 0.07 for neurophysin I and a value of 4.79 ± 0.11 for neurophysin II. Radioimmunoassay revealed barely detectable levels of adrenocorticotropin-like material in neurophysin I (12 pg/100 μg of neurophysin) and no detectable levels in neurophysin II. Both proteins were devoid of avian vasodepressor activity in the conscious chicken, melanotropic activity in vitro in frog skin, and did not effect electrolyte excretion in hydropenic rats.     

Influence of neurophysin residues 1-8 on the optical activity of neurophysin-peptide complexes. Direct evidence that the 1-8 sequence alters the environment of bound peptide

Circular dichroism was used to compare the environment of peptides bound to native and des 1-8 neurophysin in order to further elucidate the role of the neurophysin 1-8 sequence in peptide-binding. A very large positive ellipticity (approximately 6000 deg cm2 dmol-1), shown earlier to be induced in tyrosine at position 2 of peptides bound to the native protein, was determined by the present study to be paralleled by similar induced changes in tyrosine at peptide position 1. Deletion of the neurophysin 1-8 sequence led to loss of half of the induced optical activity at peptide positions 1 and 2 and changes in binding-induced optical activity in the protein, the latter partially assignable to protein disulfides. In the mononitrated native and des 1-8 proteins, the optical activity of neurophysin Tyr-49, a residue at the peptide-binding site, was reduced by 80% in complexes of the des 1-8 protein relative to those of the native protein. The results suggest a role for neurophysin Arg-8 in modulating the optical activity at the binding site by directly placing a charge proximal to the binding site and/or by altering binding site conformation. The data provide the first unambiguous evidence of a difference in the environment of bound peptide between the native and des 1-8 proteins.     

Immunochemical evidence for the transport of neurophysin in the hypothalamo-neurohypophysial system of the dog

Summary 1. An immunohistochemical study has been made of the hypothalamo-neurohypophyseal system of the dog, 20h after crushing the pituitary stalk.2. By use of a cross-species-reactive neurophysin antiserum it was shown that neurophysin is a component of the axons which originate in the supraoptic and paraventricular nuclei and terminate around blood vessels in the posterior pituitary.3. Neurophysin specific fluorescence accumulated in axons proximal to the constriction but was absent from the axons immediately distal to the site of injury.4. In dogs left for six days it was shown by radioimmunoassay that the amount of neurophysin in the hypothalamus and stalk proximal to the constriction increased twofold while that remaining in the posterior pituitary and stalk distal to the constriction decreased five-fold over the same period.5. The results are interpreted as evidence for a rapid axonal transport of neurophysin from its site of synthesis in the cell bodies of the hypothalamus to the posterior pituitary. Acknowledgements: This work was supported by a research grant to D. B. Hope from the Medical Research Council. L. O. Uttenthal was supported by a Medical Research Training Award and B. G. Livett by a Nuffield Dominions Trust Demonstratorship (Australia). We thank Mrs. Marion Martin for radioimmunoassay of neurophysin, Miss Wendy Jones for technical assistance and the U.C.L.A. Brain Information Service for help with the bibliography.     

Electron microscope immunohistochemical localization of neurophysin in the rat with hereditary diabetes insipidus

In order to study the subcellular distribution of neurophysin in the rat with hypothalamic hereditary diabetes insipidus (DI), an immunoelectron microscopic localization of neurophysin was performed in the hypothalamo-neurohypophysial system of both homozygous and heterozygous DI rats. Whereas in control rats neurophysin was localized in the granules present in the secretory neurons, in the homozygous DI rats neurophysin was found in the granules and outside the granules in the perikarya and axons of neurons of both supraoptic and paraventricular nuclei. In the heterozygous DI rats findings similar to those observed in homozygous DI rats were observed, although in the posterior pituitary, the exgranular material appeared to be less abundant than in homozygous DI rats. These results clearly demonstrated that in hyperstimulated neurons neurophysin was distributed in both granular and extragranular compartments.     

Influence of opioid peptides on release from vasopressin and oxytocin neurones of the hypothalamoneurohypophyseal system: effects of naloxone in the conscious rat

We examined the effects of acute and chronic treatments with naloxone on release of vasopressin and oxytocin from the hypothalamoneurohypophyseal system (HNS) in conscious, chronically instrumented Long-Evans rats. Plasma concentrations of vasopressin-associated neurophysin and oxytocin-associated neurophysin were evaluated before and during an intravenous infusion of 18% saline at 100 microL.kg-1 body weight.min-1 for 60 min. Acute treatment with naloxone (2.75 mumol/kg, intravenous) did not measurably alter basal plasma osmolality or vasopressin-associated neurophysin concentration, but it caused a three-fold rise in basal plasma oxytocin-associated neurophysin concentration (16 +/- 2 to 46 +/- 3 fmol/mL, p less than 0.005). Chronic treatment with naloxone (13.75 mumol/day, subcutaneous pellets) increased plasma osmolality (292 +/- 1 to 300 +/- 2 mosmol/kg H2O, p less than 0.01) by day 5, but it had no measurable effects on basal vasopressin- or oxytocin-associated neurophysin concentration. There were also no significant differences in plasma sodium concentration (144.8 +/- 1.1 vs. 142.2 +/- 1.4 mequiv./L) under both conditions. Acute and chronic treatments with naloxone accompanied by salt loading produced a five- and four-fold decrease in the rates that plasma concentration of vasopressin-associated neurophysin changed with plasma osmolality, compared with untreated salt-loaded control rats. For oxytocin secretion from the HNS, both treatments accompanied by salt loading substantially decreased the threshold for changes in relation to plasma osmolality; the rise in plasma concentration of oxytocin-associated neurophysin was similar at all levels of hyperosmotic stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions between a progestational derivative (norethisterone acetate) and ethinyl estradiol on urinary elimination of neurophysin, ocytocin, and vasopressin and on the blood level of neurophysin I and II in normal human beings

Neurophysin 1 (a peptide associated with antidiuretic homone, ADH), ADH, and oxytocin were measured in serum or urine of 3 men who took 10 mcg ethinyl estradiol with 5 mg norethisterone acetate twice daily for 6 days and in 3 who took the estrogen only. Neurophysin in plasma was determined by radioimmunoassy, and individually by starch gel electrophoresis, extraction, and assay. Total urine neurophysin was measured by radioimmunoassy. ADH and oxytocin were quantitated by column chromatography and radioimmunoassy. With both steroids, there was no change in neurophysin, ADH, or oxytocin. Ethinyl estradiol alone increased urinary neurophysin from 112.3 to 595 ng/hour (n.s.), oxytocin from 41-132 ng/hour (p less than .005), and ADH from 9.7-57.2 ng/hour (p less than .01). As in pregnant women compared to normal women, estrogen treatment stimulated plasma neurophysin 1 more than neurophysin 2 in these subjects. These results confirm those found in rats.     

PROTEIN-CARBOXYL METHYLTRANSFERASE OF THE BOVINE POSTERIOR PITUITARY GLAND: NEUROPHYSIN AS A POTENTIAL ENDOGENOUS SUBSTRATE

Abstract— The kinetics of methylation of neurophysin by the enzyme protein-carboxyl methyltransferase (EC 2.1.1.24) was investigated, together with the subcellular distribution of enzyme and endogenous substrate in the bovine posterior pituitary gland. Using a preparation of the methyltransferase, purified from acetone-dried powders of bovine posterior pituitary gland, bovine neurophysin was found to be a better substrate than those proteins usually employed, neurophysin having a lower apparent K _m and higher V _max than the other substrates. Arginine-vasopressin did not inhibit the methylation of neurophysin, supporting the hypothesis that methylation and hormone-binding sites are different. Vasopressin, however, increased the rate of methylation of neurophysin I by the methyltransferase. but was without effect on the rates of methylation of the other components of bovine neurophysin. After subcellular fractionation of fresh bovine posterior pituitary glands, some 77% of the recovered methyltransferase activity was found in the high-speed supernatant fraction, being distributed throughout all the fractions similarly to lactate dehydrogenase. Neurophysin is therefore unlikely to become a substrate until released from the neurosecretory granules during stimulation of the gland. Some evidence is presented for the presence of an endogenous substrate other than neurophysin, in the high-speed supernatant fraction.     

Evidence for the secretion of immunoreactive neurophysin I in addition to oxytocin from the ovary in cattle

In Exp. I, blood samples were collected simultaneously from the posterior vena cava and jugular vein or aorta from 7 heifers every 5-20 min for 2-5 h. Concomitant pulsatile secretion of oxytocin and immunoreactive neurophysin I was detected in the vena cava, but not in the jugular vein or aorta. Concentrations of oxytocin and immunoreactive neurophysin increased earlier and were higher in the vena cava than in the jugular vein or aorta after the injection of a luteolytic dose of prostaglandin F-2 alpha analogue during the mid-luteal phase of the oestrous cycle, demonstrating its ovarian but not pituitary origin. In Exp. II, blood samples were collected from the jugular vein every 12 h during 1 week after oestrus. Follicular growth had been stimulated during the preceding oestrous cycle with PMSG (10 heifers and cows) or with FSH (5 animals); 6 heifers served as controls. There was a high correlation between the number of follicles or CL and the increase in oxytocin and immunoreactive neurophysin I. Although PMSG had a greater luteotrophic effect than did FSH on progesterone secretion, a similar stimulation of oxytocin and immunoreactive neurophysin I was not observed. It is concluded that immunoreactive neurophysin I and oxytocin are secreted from the ovary in concentrations dependent upon the number of corpora lutea (and of follicles) present. During the mid-luteal period the secretion occurs in a concomitant pulsatile fashion.     

Structural requirements of peptide hormone binding for peptide-potentiated self-association of bovine neurophysin II

Site-specific, truncated, and sequence-simplified analogs of the hormone [Arg8]vasopressin were investigated for the relationship between their abilities to recognize immobilized bovine neurophysin and to promote neurophysin self-association. Peptide binding to neurophysin was measured quantitatively by analytical high performance affinity chromatography on immobilized bovine neurophysin II. Neurophysin self-association, measured as binding of soluble to immobilized neurophysin, was promoted (made higher affinity) by soluble peptide hormone and its analogs, with the effect of particular peptides being proportional to their binding affinities for neurophysin. Sequence-redesigned peptides able to recognize neurophysin, including dipeptide amides, were able to potentiate the self-association to the same extent as the natural hormone when tested at concentrations adjusted to effect equal degrees of saturation of neurophysin. The relationship between peptide affinity to neurophysin and the potentiation of self-association suggests that the latter is directly dependent on the former and can occur even with limited segments of hormone sequence. The data fit best to a model in which hormone binding and self-association surfaces of neurophysin are separate and linked through the neurophysin molecule to produce cooperativity (hormone-promoted self-association). Given that only limited structural elements of hormone are required for promoting self-association, the results fit less well with models in which cooperativity requires that hormone make dimer-stabilizing contacts with both self-associating subunits of neurophysin simultaneously.     

Demonstration of neurophysin in the hypothalamo-neurohypophysial system of the normal and dehydrated rat by the use of cross-species reactive anti-neurophysins

Summary Antiserum raised against porcine neurophysin-II and ovine neurophysin-III has been shown to cross react with the three neurophysins of the rat. The antisera were used in the immunohistological demonstration of neurophysin in the posterior pituitary gland and the hypothalamic nuclei of a normal rat and a rat subjected to varying degrees of osmotic stress. Depletion of neurosecretory material from the neural lobe of the stimulated rat is also accompanied by a concomitant reduction in the neurophysin and vasopressin content of the gland. Neurophysin is present in the cytoplasm of both the supraoptic and paraventricular nuclei.This work was financed by the Zealand Medical Research Council and the Auckland Medical Research Foundation. We thank Miss V. Bailey for help with the photographs.