Design of a photoaffinity label for the hormone binding site of neurophysin.

The photolabile peptide, L-methionyl-L-tyrosyl-p-azido-L-phenylalaninamide, was synthesized by solution methods. This peptide, as well as the analogous species containing tritiated methionine, were found to bind reversibly and specifically, in the dark, to bovine neurophysin II. The dissociation constant, stoichiometry, and pH-dependence of this noncovalent interaction are typical of those properties for hormone (oxytocin) and hormone-like ligand binding to neurophysin II. Under photolytic conditions, methionyl-tyrosyl-p-azidophenylalaninamide causes irreversible inhibition of the noncovalent ligand binding activity of neurophysin II. This inactivation was achieved to the extent of about 90%. Both the dark and light (photolytic) interactions of the photolabile peptide with neurophysin II indicate its reaction at the hormone binding site of the protein and thus its potential use to identify amino acid residues at this site by covalent photoaffinity labelling.     

High-performance liquid chromatography and studies of neurophysin—neurohypophysial hormone pathways

High-performance liquid chromatography (HPLC) is being used extensively to characterize active polypeptides, precursor processing mechanisms, and cooperative peptide—protein noncovalent complexes in neuroendocrine pathways for neurohypophysial peptide hormones, oxytocin and vasopressin, and the hormone-associated proteins, neurophysins. Reversed-phase and ion-exchange HPLC polypeptide mapping have been used to detect the hormones, associated proteins, and other molecular forms containing these. This mapping but also ultimately to identify anatomical sites which contain the neurophysin/ hormone molecular pathways and to define the relatedness of polypeptide forms contained in different pathways. Reversed-phase HPLC also has provided a means to study proteolytic precursor processing, both to isolate synthetic and semisynthetic polypeptides and intermediates produced by these reactions. Finally, bioaffinity HPLC is being evaluated as a separatory and analytical tool. The latter includes its use to characterize the noncovalent peptide—protein and protein—protein interactions which occur among the molecular forms of the neurophysin/hormone pathways. These experiments typify the impact of HPLC for both analytical and preparative separations in studies of biologically active peptides and proteins.     

Immunocytochemical identification of dynorphin-containing vesicles in Brattleboro rats

Vasopressin and its carrier protein, vasopressin-associated neurophysin, are co-packaged together with an opioid peptide, dynorphin, into 160 nm diameter neurosecretory vesicles in the normal rat hypothalamo-neurohypophysial system. The homozygous Brattleboro rat lacks vasopressin and vasopressin-associated neurophysin, but contains substantial amounts of dynorphin in the vasopressin-deficient neurosecretory cells. We used post-embedding electron microscopic immunocytochemistry to determine the subcellular location of dynorphin in Brattleboro rats. The results show that dynorphin is present within 100 nm neurosecretory vesicles in homozygous Brattleboro cell bodies and axons, and within 160 nm vesicles in heterozygous (control) neurosecretory cell bodies and axons. Oxytocin-associated neurophysin is present in a separate population of magnocellular neurons in both homozygous and heterozygous rats, and is contained within 160 nm vesicles in both cases. Therefore, the absence of synthesis of the vasopressin prohormone results in a dramatic reduction of neurosecretory vesicle size, despite the continued synthesis and packaging of dynorphin peptides.     

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.     

Structure–Function Relationships of the Vasopressin Prohormone Domains

1. In this review the structure–function relationships of the different vasopressin prohormone domains are dated and discussed, with special reference to the neurophysin and glycopeptide domains.2. The primary structures of the currently known neurophysins and glycopeptide sequences are compared and discussed.3. The hormone-binding and aggregational properties of neurophysin are reviewed and related to a possible function within the regulated secretory pathway.4. It is proposed, based on the properties reviewed here as well as our own data shown here, that the sorting of the vasopressin prohormone is initiated by hormone binding, which triggers aggregation of the prohormone into the characteristic dense cores of the regulated secretory pathway.5. This may suggest that prohormone sorting into the regulated secretory pathway is, in general, determined by noncovalent, intramolecular interactions that promote aggregation.     

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.     

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.     

Cytophysiology of neurosecretory axon terminals in the brain of an annelid (Ophryotrocha puerilis,Polychaeta)

Summary In the posterior part of the brain of the protandric polychaete Ophryotrocha puerilis neurosecretory cells form prominent axon terminals. The terminals are arranged in two complexes. The perikarya of these presumably monopolar neurons are scattered in the anterior part of the cerebral perikaryal layer. In females the terminals store large amounts of neurosecretory material. It has been suggested earlier that neurosecretions of the terminals may play a role during sex reversal from females to males. Application of histamine caused the release of neurosecretory material from the respective terminals in females. However, this discharge was not followed by sex reversal. Application of reserpine had no influence on the terminals. Neither by in vivo observation nor by ultrastructural analysis any effect of reserpine on the terminal complexes could be observed. In isolated terminals filled with neurosecretory material from females, catecholamines could not be detected by HPLC. Also, polyclonal antibodies against dopamine did not stain the terminal complexes. Furthermore, the complexes did not develop any fluorescence after glyoxylic acid treatment. Therefore, the present results contradict the hypothesis that the neurosecretory material of the respective axon terminals is catecholaminergic and that it is involved in sex differentiation. The function of the secretory neurons studied here remains unclear.Abbreviations AT axon terminal - CA catecholamine(s) - DA dopamine - DAB diaminobenzidine - GA glyoxylic acid - GIF glyoxylic acid-induced fluorescence - LY Lucifer Yellow - MB methylene blue - NSM neurosecretory material - OTH ootropic hormone - TC terminal complex     

Ultrastructural and cytochemical features of SV 40 transformed hypothalamic cell lines

Summary A continuous cell line was previously obtained by Simian Virus (SV) 40 transformation of primary cultures of dissociated mouse fetal hypothalami. One clone from this cell line has been previously shown to possess some of the ultrastructural features, immunological properties and synthesizing capacities of magnocellular hypothalamic neurons which secrete vasopressin and neurophysins. The present paper reports on the morphological characterization of 14 other clones or subclones of the original cell line, using the following criteria: phase contrast microscopy, electron microscopy, Gomori's aldehyde fuchsin staining, cytochemical detection of -glucuronidase, immunochemical staining with antisera against bovine neurophysin I, bovine neurophysin II, lys-vasopressin, oxytocin, LH-RH and TRH.The results allowed the conclusion that the clones as well as the subclones can be distributed into two groups: 1) neurosecretory neurons which all possess several of the ultrastructural and cytochemical features of the neurophysin-vasopressin synthesizing clone, and 2) primitive nerve cells which are devoid of such features but display numerous bundles of filaments. In addition some clones were found to display intermediate features between groups 1 and 2. A similar diversity was observed within clones of the original strain and subclones of a neurosecretory clone. It is suggested that the primitive clones could represent precursors of the neurosecretory clones.This work is dedicated to Professor W. Bargmann in honour of his 70th birthday     

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.     

Functional epitopes for site 1 of human prolactin

Human prolactin (hPRL) binds two human prolactin receptor molecules, creating active heterotrimeric complexes. Receptors bind dissimilar hormone surfaces termed site 1 and site 2 in an obligate ordered process. We sought to map the functional epitopes in site 1 of hPRL. Extensive alanine mutagenesis (102 of the 199 residues) showed approximately 40% of these mutant hPRLs changed the ΔG for site 1 receptor binding. Six of these residues are within 3.5 ? of the receptor and form the site 1 functional epitopes. We identified a set of noncovalent interactions between these six residues and the receptor. We identified a second group of site 1 residues that are between 3.5 and 5 ? from the receptor where alanine mutations reduced the affinity. This second group has noncovalent interactions with other hormone residues and stabilized the topology of the functional epitopes by linking these to the body of the protein. Finally, we identified a third group of residues that are outside site 1 (>5 ?) and extend to site 2 and whose mutation to alanine significantly weakened receptor binding at site 1 of prolactin. These three groups of residues form a contiguous structural motif between sites 1 and 2 of human prolactin and may constitute structural features that functionally couple sites 1 and 2. This work identifies the residues that form the functional epitopes for site 1 of human prolactin and also identifies a set of residues that support the concept that sites 1 and 2 are functionally coupled by an allosteric mechanism.     

Binding of oxytocin and 8-arginine-vasopressin to neurophysin studied by 15N NMR using magnetization transfer and indirect detection via protons

NMR was used to monitor the binding to neurophysin of oxytocin and 8-arginine-vasopressin, 15N labeling being used to identify specific backbone 15N and 1H signals. The most significant effects of binding were large downfield shifts in the amino nitrogen resonance of Phe-3 of vasopressin and in its associated proton, providing evidence that the peptide bond between residues 2 and 3 of the hormones is hydrogen-bonded to the protein within hormone-neurophysin complexes. Suggestive evidence of hydrogen bonding of the amino nitrogen of Tyr-2 was also obtained in the form of decreased proton exchange rates on binding; however, the chemical shift changes of this nitrogen and its associated proton indicated that such hydrogen bonding, if present, is probably weak. Shifts in the amino nitrogen of Asn-5 and in the -NH protons of both Asn-5 and Cys-6 demonstrated that these residues are significantly perturbed by binding, suggesting conformational changes of the ring on binding and/or the presence of binding sites on the hormone outside the 1-3 region. No support was obtained for the thesis that there is a significant second binding site for vasopressin on each neurophysin chain. The behavior of both oxytocin and vasopressin on binding was consistent with formation of 1:1 complexes in slow exchange with the free state under most pH conditions. At low pH there was evidence of an increased exchange rate. Additionally, broadening of 15N resonances in the bound state at low pH occurred without a corresponding change in the resonances of equilibrating free hormone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence studies of native and modified neurophysins. Effects of peptides and pH.

The effect of neurophysin-hormone interaction on the environment of the single tyrosine of bovine neurophysin (Tyr-49) and on that of the tyrosine of oxytocin and vasopressin was studied by fluorescence; tyrosine-free peptides were used to determine effects on Tyr-49, and acetylated neurophysin was used to determine effects on the hormone tyrosine. Binding increases the fluorescence intensity of Tyr-49 by 130% while the fluorescence of the hormone tyrosine is almost completely quenched. Correlation of these results with those obtained on binding oxytocin or vasopressin to native neurophysin indicates that in the hormone complexes less than half of the fluorescence of Tyr-49 is lost by F?rster energy transfer to the quenched hormone tyrosine. These results support spin-label studies in indicating that the distance between Tyr-49 and the tyrosine of hormone bound to the strong hormone binding site is greater than 5 A. In the absence of peptides, the fluorescence of Tyr-49 increases by 40% on lowering the pH from 6.2 to 2. Titration of the acid fluorescence transition in bovine neurophysins-I and -II, and in bovine neurophysin-II treated with carboxypeptidase B to remove the Arg-Arg-Val sequence at the carboxyl terminus, indicates that this transition is due to titration of a side-chain carboxyl with an intrinsic pK of 4.6. The effects of guanidine, glycerol, and disulfide cleavage on the magnitude of the acid transition indicate that the conformational information necessary for the transition resides within the amino acid sequence adjacent to Tyr-49. Accordingly, the fluorescence acid transition is attributed to decreased quenching by Glu-46 or Glu-47 upon protonation. Glycerol is shown to perturb the glutamate-tyrosine interaction in the absence of general conformational effects. Comparison of the fluorescence low-pH transition with that of the low-pH circular dichroism transition of nitrated neurophysins suggests that the fluorescence and CD transitions reflect related, but not necessarily identical, phenomena. In an appendix, evidence is presented which suggests that the products of carboxy-peptidase digestion of bovine neurophysin-II are the same as two minor bovine neurophysin components, one of which is neurophysin-C.     

Ultrastructural immunocytochemical characterization of isotocin,vasotocin and neurophysin neurons in the magnocellular preoptic nucleus of the goldfish

Summary We describe the ultrastructural localization of isotocin, vasotocin and neurophysin in the magnocellular preoptic nucleus of the goldfish. With the aid of immunocytochemical techniques, we see staining both in classical neurosecretory granules and in diffuse agranular form throughout somata and processes. Signs of cellular and synaptic interactions between chemically identified neurons include axon terminals which contain vasotocin immunoreactivity and membrane specializations (puncta adhaerentia) between adjacent somata. Our investigations provide an anatomical basis for neuroendocrine and neurotransmitter-like functions of peptidergic neurons in the teleost preoptic nucleus.     

Localization of hormone secreting pathways in the brain by immunohistochemistry and light microscopy: a review.

Immunocytochemical techniques are now being used to localize hypothalamic neurosecretory hormones and related peptides in the mammalian brain. The data are probably incomplete, due primarily to false negative results. A number of previous assumptions concerning these pathways have been confirmed while other unexpected results were obtained. As expected, vasopressin and oxytocin and their associated proteins, neurophysins, were found in the magnocellular cell bodies of the hypothalamus and in their axonal projections to the neural lobe of the pituitary. Gonadotropin-releasing hormone (Gn-RH), somatostatin, and thyrotropin-releasing hormone (TRH) were located in what appears to be parvicellular nerve terminals on portal capillaries. Gn-RH has been found in perikarya in the arcuate nucleus, which is considered a source of fibers to the portal capillary bed. An extensive network of cell bodies and fibers in the preoptic area was also found to contain Gn-RH, and others in the periventricular nucleus in the anterior hypothalamus reacted with antiserum to somatostatin. Unexpected was considerable evidence that vasopressin is secreted directly into hypophyseal portal blood. This hormone and its neurophysin were also found in parvicellular neurons in the suprachiasmatic nucleus of rodents. All the hormones were found in fibers in the organum vasculosum of the lamina terminalis and in the posterior pituitary gland.     

The Clinical Physiology of Water Metabolism: Part I: The Physiologic Regulation of Arginine Vasopressin Secretion and Thirst

Water balance is tightly regulated within a tolerance of less than 1 percent by a physiologic control system located in the hypothalamus. Body water homeostasis is achieved by balancing renal and nonrenal water losses with appropriate water intake. The major stimulus to thirst is increased osmolality of body fluids as perceived by osmoreceptors in the anteroventral hypothalamus. Hypovolemia also has an important effect on thirst which is mediated by arterial baroreceptors and by the renin-angiotensin system. Renal water loss is determined by the circulating level of the antidiuretic hormone, arginine vasopressin (AVP). AVP is synthesized in specialized neurosecretory cells located in the supraoptic and paraventricular nuclei in the hypothalamus and is transported in neurosecretory granules down elongated axons to the posterior pituitary. Depolarization of the neurosecretory neurons results in the exocytosis of the granules and the release of AVP and its carrier protein (neurophysin) into the circulation. AVP is secreted in response to a wide variety of stimuli. Change in body fluid osmolality is the most potent factor affecting AVP secretion, but hypovolemia, the renin-angiotensin system, hypoxia, hypercapnia, hyperthermia and pain also have important effects. Many drugs have been shown to stimulate the release of AVP as well. Small changes in plasma AVP concentration of from 0.5 to 4 μU per ml have major effects on urine osmolality and renal water handling.     

Localization of neurophysin in the rat supraoptic nucleus

Summary Localization of neurophysin in neurons of the supraoptic nucleus was accomplished using an unlabeled-antibody, post-embedding, immunoperoxidase technique. Neurophysin was exclusively associated with neurosecretory granules within cell bodies of supraoptic neurons and their processes.Supported by U.S.P.H.S. Grant HD-08867     

Leucokinin and diuretic hormone immunoreactivity of neurons in the tobacco hornworm,Manduca sexta,and co-localization of this immunoreactivity in lateral neurosecretory cells of abdominal ganglia

Because leucokinins stimulate diuresis in some insects, we wished to identify the neurosecretory cells in Manduca sexta that might be a source of leucokinin-like neurohormones. Immunostaining was done at various stages of development, using an antiserum to leucokinin IV. Bilateral pairs of neurosecretory cells in abdominal ganglia 3–7 of larvae and adults are immunoreactive; these cells project via the ipsilateral ventral nerves to the neurohemal transverse nerves. The immunoreactivity and size of these lateral cells greatly increases in the pharate adult, and this change appears to be related to a period of intensive diuresis occurring a few days before adult eclosion. Relationships of these neurons to cells that are immunoreactive to a M. sexta diuretic hormone were also investigated. Diuretic hormone and leucokinin immunoreactivity are co-localized in the lateral neurosecretory cells and their neurohemal projections. A median pair of leucokinin-immunoreactive, and a lateral pair of diuretic hormone-immunoreactive neurons in the larval terminal abdominal ganglion project to neurohemal release sites within the cryptonephridium. The immunoreactivity of these cells is lost as the cryptonephridium is eliminated during metamorphosis. This loss appears to be related to the change from the larval to adult pattern of diuresis.     

Bovine neurophysin lipid complex. Their isolation, characterization and reaggregation

A lipid-containing neurophysin fraction was isolated and purified from bovine posterior pituitary glands by acid extraction and affinity chromatography on a heparin-Sepharose 4B column. This lipid-rich fraction was found to be composed of noncovalent aggregates of neurophysin proteins and phospholipids such as phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine and sphingomyelin. The lipid-containing neuophysin was delipidated by treatment with choloform-methanol. The resultant apoproteins were characterized as bovine neuroions were developed for the reaggregation of purified bovine neurophysin-I and -II with lipids extracted from bovine posterior pituitary and hypothalamus and with synthetic lecithin. The resultant neurophysin lipid complexes have been shown to band upon isopycnic centrifugation at densities different from those of the respective purified bovine neurophysins.