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PubMed1988

Gonadal steroids influence neurophysin II distribution in the forebrain of normal and mutant mice.

The distribution of arginine vasopressin-associated neurophysin (neurophysin II) immunoreactivity was investigated in normal and mutant house mice during development and after various gonadal steroid manipulations. During postnatal development of normal mice dense networks of neurophysin II immunoreactivity in the lateral septal nucleus and lateral habenular nucleus appeared earlier in male than in female mice, with an adult pattern of immunoreactivity being attained by 8 weeks and 12 weeks of age, respectively. The neurophysin II immunoreactivity in the male was denser than that in female mice. After gonadectomy of adult normal mice there was a gradual loss of neurophysin II immunoreactivity in the lateral septum and lateral habenula over a period of 15 weeks. In hypogonadal mice, a mutant in which gonadal development is arrested postnatally due to a deficiency in hypothalamic gonadotrophin releasing hormone, no immunoreactive neurophysin II could be detected in the lateral septum or lateral habenula. A pattern of neurophysin II immunoreactivity similar to that in normal control mice was observed in hypogonadal mice which had been implanted for 4 weeks with silicone elastomer capsules containing testosterone or oestradiol-17 beta, but not 5 alpha-dihydrotestosterone or progesterone. Stimulation of gonadal development and endogenous steroid production in hypogonadal mice by third ventricular grafts of preoptic area tissue from normal neonatal animals also produced a normal pattern of neurophysin II immunoreactivity in the lateral septum and lateral habenula. In the androgen-insensitive testicular feminized mouse immunoreactive neurophysin II was undetectable in the lateral septum and lateral habenula. Treatment of testicular feminized mice with oestradiol-17 beta, but not progesterone, produced a normal pattern of neurophysin II immunoreactivity. The main immunohistological findings were confirmed by radioimmunoassay of tissue extracts which showed that the concentration of arginine vasopressin in lateral septum was far greater in normal males than females and was undetectable in hypogonadal mice; no oxytocin could be detected in the septum of normal or hypogonadal mice. These results show that the expression of neurophysin II immunoreactivity in the lateral septum and lateral habenula of the mouse brain is dependent on the presence of aromatizeable androgens or oestrogens.

Aging↗
PubMed1980

Immunological identification of high molecular weight forms common to bovine neurophysin and vasopressin.

Extracts of bovine neurohypophysis made in acid/ethanol solution containing protease inhibitors were fractionated by two successive filtrations on Sephadex G-75 columns equilibrated in the presence and then in the absence of 4 M urea. Analysis of the pattern of neurophysin-like immunoreactivity in the eluate, with two different antibodies, indicated the presence of high M(r) forms of neurophysin (apparent sizes, [unk]70,000 and 20,000-25,000, respectively) besides the M(r) 10,000 neurophysin. [8-Arginine]vasopressin-like immunoreactivity was also detected, coeluting with the neurophysin-like species, in the material recovered in the exclusion and M(r) 20,000-25,000 elution volumes of the same molecular sieve fractionation of neurohypophyseal extracts. Upon subsequent Sephadex G-150 filtration, the immunoreactive material recovered in the exclusion volume of the Sephadex G-75 filtration showed an apparent M(r) of approximately 140,000. Both neurophysin-like and vasopressin-like immunoreactivities coeluted in the same volume. The elution profile of this M(r) 140,000 material was unmodified when reanalyzed by the same molecular sieve filtration after exposure to 8 M urea. When these M(r) 140,000 immunoreactive forms of vasopressin and neurophysin were submitted to affinity chromatography on anti-neurophysin antibodies immobilized on Sepharose, both immunoreactivities were selectively coadsorbed to the immunoadsorbent. Similarly, the neurophysin and vasopressin immunoreactivities associated with M(r) approximately 25,000 were retained together on the same anti-neurophysin immunoadsorbent. The M(r) 140,000 and M(r) 25,000 species having both neurophysin and [8-arginine]vasopressin antigenic determinants generated the two neurosecretory components when exposed to proteolytic activities. This in vitro processing was inhibited in acid medium, at low temperature, and in the presence of a mixture of protease inhibitors. It is concluded that these two large forms of proteins containing both neurophysin and vasopressin may represent common biosynthetic precursors of these two neurohypophyseal components.

Animals↗
PubMed1977

The physiological release of specific individual neurophysins into the circulation of pigs.

Specific homologous radioimmunoassays for the two major porcine neurophysins have been developed and utilized to measure plasma neurophysins during events known to release vasopressin (dehydration and hemorrhage) and oxytocin (parturition and suckling). During hemorrhage plasma neurophysin I increased 2-25 times and decreased toward basal levels after reinfusion of the blood while plasma neurophysin II was low and showed only minor fluctuations. Neurophysin II was released during parturition and suckling in a pattern similar to that reported for oxytocin release during these events. A rise in plasma neurophysin II occurred towards the end of parturition and spurt release occurred in suckling. The function of neurophysins in the plasma is unknown but porcine neurophysin I has been shown to be released independently into the circulation in response to hemorrhage. Independent release of neurophysin II during parturition and suckling was not demonstrated. In the pig, release of neurophysin I may be associated with vasopressin release and neurophysin II associated with oxytocin release.

Animals↗
PubMed1977

Presence of two neurophysins in the human pineal gland.

The tentative identification of two neurophysins in human pineal glands is reported. The presence of these carrier-proteins for neurohormones was demonstrated by two different radioimmunossays: one highly specific for human pituitary estrogen-stimulated neurophysin (h-ESN) and the second for pituitary bovine neurophysin II (b-NII). These neurophysins accounted for about 0.425% of the total soluble pineal proteins. They were eluted from Sephadex G-75 column with the same elution volume as 125I labeled h-ESN. Their dilution curves were parallel to the standard curves of h-ESN and b-NII, respectively. Electrophoresis on polyacrylamide gel separated two distinct neurophysins. The less anodic neurophysin corresponding to b-NII and the faster moving one to h-ESN. The two proteins were named human pineal neurophysins I and II (h-PNI and h-PNII) in the order of their electrophoretic mobility. Purification of pineal neurophysins by isoelectric focusing resulted also in the separation of two neurophysins: one, recognized by antibody against h-ESN with an isoelectric point (pI) of 4.6, the other recognized by antibody against b-NII with a pI of 4.9. The presence of neurohormone(s), assessed by radioimmunoassay for [8-arginine] vasopressin, was also demonstrated. Under optimal conditions for the association the neurophysin and the neurohormone were eluted from Sephadex columns as a complex. This complex could be dissociated in 0.1N formic acid and the neurophysin and the neurohormone separated on Sephadex G-75.

Chromatography, Gel↗
PubMed1981

Oxytocin in human plasma: correlation with neurophysin and stimulation with estrogen.

RIA for the measurement of oxytocin in human plasma is described. Extraction of oxytocin from larger peptides in plasma used acetone precipitation with a 75% +/- 2 SEM recovery of oxytocin. Nonspecific binding of the assay was less than 4%, and the minimum level of detection was 0.2 microunits/tube. No cross-reactivity was noted with neurophysins, arginine, or lysine vasopressin. The mean basal level (+/- SEM) of oxytocin in men was 1.80 +/- 0.07 microunits/ml and was not different in normal women (1.71 +/- 0.07 microunits/ml). Changes in posture had no effect on the levels of oxytocin. Samples obtained every 15 min over 4 h showed no pulsatile secretion of oxytocin. In women chronically receiving estrogen as an oral contraceptive, oxytocin was greater than normal, (4.59 +/- 0.51 microunits/ml; P less than 0.01). Estrogen-stimulated neurophysin was also elevated (8.45 +/- 1.99 ng/ml; P less than 0.005). Acute ingestion of estrogen caused an increase in the level of oxytocin in plasma by 12 h and a concomitant elevation of estrogen-stimulated neurophysin. When the neurophysin was isolated from plasma obtained from a subject after ingestion of estrogen, the neurophysin from plasma comigrated on a polyacrylamide gel with a human pituitary standard of estrogen-stimulated neurophysin. In the studies in which neurophysin was elevated, the correlation between the level of oxytocin and the level of estrogen-stimulated neurophysin in plasma was significant (P less than 0.01). The observation that estrogen administration stimulates the release of oxytocin and estrogen-stimulated neurophysin provides additional evidence that this neurophysin is the oxytocin-neurophysin of man.

Animals↗
PubMed1988

[Common precursors of neurohypophysial hormones and neurophysins].

Neurohypophysial hormones and neurophysins are former domains of common precursors processed during the axonal transport from hypothalamus to neurohypophysis. Two neurohormones, an oxytocin-like and a vasopressin-like, and two neurophysins, termed VLDV- and MSEL-neurophysins according to residues in positions 2, 3, 6 and 7, are usually found in vertebrate species. In mammals, a non-covalent stoichiometric and reversible complex including the two neurohormones and the two neurophysins has been isolated. In contrast to other mammals investigated, the three-domain precursor of vasopressin (vasopressin, MSEL-neurophysin and copeptin) is not completely processed in guinea pig and an intermediate precursor including MSEL-neurophysin and copeptin linked by an arginine residue has been isolated and sequenced. "In vitro" processing of this intermediate through trypsin-Sepharose has revealed cleavages only in the inter-domain region, showing the role of precursor conformation in the processing. In neurosecretory granules from guinea pig, only free vasopressin and MSEL-neurophysin have been detected. In bovine foetus at the age of 3 and 7 months, only vasopressin and oxytocin in molar ratios 4 and 3, respectively, have been identified as well as adult MSEL- and VLDV-neurophysins. No vasotocin and no additional neurophysin when compared to the adult have been found. Diabetes insipidus rats from the Brattleboro strain have been examined in order to identify an abnormal vasopressin precursor. No free vasopressin and no free MSEL-neurophysin have been detected through high pressure liquid chromatography whereas oxytocin and VLDV-neurophysin have been identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1975

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.

Animals↗
PubMed1979

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 Förster 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.

Animals↗
PubMed1975

Isolation, assay, and secretion of individual human neurophysins.

Human neurophysin was isolated from acetone-dried human posterior pituitaries and separated into two major neurophysin peptides by ion exchange chromatography and into four major peptides by preparative disk gel electrophoresis. Antisera raised in rabbits distinguished only two specific antigenic sites on the isolated neurophysin peptides. Individual sensitive and specific radioimmunoassays for two human neurophysins were developed. These assays were used to measure each neurophysin in unextracted human plasma. The two neurophysins are secreted independently in man. One assay measures a neurphysin that is specifically secreted in response to estrogen administration, estrogen-stimulated neurophysin (ESN). The other assay measures a neurophysin that is specifically secreted in response to cigarette smoking, nicotine-stimulated neurophysin (NSN). The mean ESN is 1.1 ng/ml plus or minus 0.7 SD in women and 1.0 ng/ml plus or minus 0.7 SD in men. The mean NSN is 0.9 ng/ml plus or minus 0.2 SD in women and 0.6 ng/ml plus or minus 0.3 SD in men. It is proposed that these may prove to be a specific human "oxytocinneurophysin," ESN, and a human "vasopressin-neurophysin," NSN.

Animals↗
PubMed1968

The isolation of the native hormone-binding proteins from bovine pituitary posterior lobes. Crystallization of neurophysin-I and-II as complexes with [8-arginine]-vasopressin.

1. The native hormone-binding proteins, neurophysin-I and -II, have been isolated from acetone-desiccated bovine pituitary posterior lobes. 2. Neurophysin-I and -II are present in approximately equal quantities in the tissue and are localized in the neurosecretory granules. 3. The apparent molecular weight, determined by equilibrium sedimentation of neurophysin-I, was 19000 and that of neurophysin-II was 21000; their sedimentation coefficients, S(20,w), were 1.66 and 2.02s respectively. 4. Neurophysin-I and -II are similar in amino acid composition. Neurophysin-II was distinguished from neurophysin-I by the absence of histidine. 5. The proteins form complexes with oxytocin as well as with vasopressin. Complexes of both proteins with [8-arginine]-vasopressin have been crystallized. 6. Bioassay of the pressor and oxytocic activities of the crystals shows that neurophysin-I binds three molecules of either vasopressin or oxytocin whereas neurophysin-II binds only two molecules of each hormone per molecule of protein. Complexes containing two molecules of oxytocin and one molecule of [8-arginine]-vasopressin per molecule of protein are formed by neurophysin-I and -II; both proteins appear to possess three polypeptide-binding sites/molecule.

Amino Acids↗
PubMed1977

Interaction of bovine neurophysin with oxytocin and vasopressin measured by temperature-jump relaxation.

The interaction between bovine neurophysins I and II and oxytocin and vasopressin was measured using temperature-jump relaxation. A single relaxation time in the 10 to 90 ms range was noted for each solution. This time depended upon the concentration of both neurophysin and hormone and increased with increasing pH. The formation rate constants (+/- SE) for the interaction of neurophysin I dimer with the protonated form of oxytocin and vasopressin at pH 7.4 in 0.1 M KNO3 and 25 degrees C were 2.8 (+/- 0.4) x 10(6) and 2.3 (+/- 0.5) x 10(6) M-1 s-1, respectively; the dissociation rate constants were 11 and 15 s-1, respectively.For neurophysin II dimer, formation rate constants were 6.0 (+/- 0.2) x 10(6) and 2.4 (+/- 0.3) x 10(6) M-1 s-1; dissociation rate constants were 24 and 16 s-1 for oxytocin and vasopressin, respectively. Formation rate constants for the interaction of neurophysin monomer with protinated hormone were approximatley an order of magniment with circular dichorism and pH titration studies which indicate that this interaction occurs between a negatively charged carboxyl group on the neurophysin and a protonated alpha-amino group on the hormone. Our results indicate that oxytocin and vasopressin bind with greater affinity to the neurophysin dimer than the monomer and that the binding of oxytocin and vasopressin in neurophysin dimer than the monomer and that the binding of oxytocin and vasopressin in neurophysin dimer at pH 7.4 and concentrations between 10(-4) and 10(-5) M is nearly identical for each hormone.

Animals↗
PubMed1979

Immunological and chemical identification of a neurophysin-containing protein coded by messenger RNA from bovine hypothalamus.

The biosynthetic origin of the 10,000 molecular weight neurophysins, carriers of the peptide hormones oxytocin and vasopressin, has been studied by cell-free synthesis, Poly(A)-RNA was isolated from bovine hypothalamus and translated in a wheat germ system containing (35)S- or (3)H-labeled amino acids. A number of unique [(35)S]cysteine- but few [(35)S]-methionine-labeled proteins were coded by hypothalamic mRNA. A single, major, isotopically labeled protein (molecular weight 23,000-25,000) was immunoprecipitated from these translation mixtures by addition of purified antibodies against bovine neurophysin II and subsequent addition of Cowan I strain of Staphylococcus aureus. Specificity of the immunoprecipitation was demonstrated by competition with unlabeled authentic neurophysins and the absence of competition with structurally unrelated ovalbumin. Furthermore, neither nonimmune serum nor purified antibodies against ribonuclease immunoprecipitated the protein. The [(35)S]cysteine-labeled protein that was specifically immunoprecipitated was oxidized with performic acid and digested with trypsin in the presence of unlabeled, authentic bovine neurophysin II. Peptide mapping revealed that most of the major [(35)S]cysteine-labeled peptides (of the translation product) were identical to major cysteine-containing peptides of authentic neurophysin. The data show that hypothalamic mRNA directs the translation of several unique cysteine-rich proteins in an in vitro cell-free system. Furthermore, one of these proteins, which has a higher molecular weight than authentic neurophysin, is recognized by purified antibodies to bovine neurophysin II and has cysteine-containing tryptic peptides in common with those of authentic neurophysin. The data suggest that this protein is the primary translation product, pre-pro-neurophysin.

Animals↗
PubMed1987

Enzymatic protein carboxyl methylation in rat posterior pituitary: neurophysins in rapid-turnover pool determine methyl accepting capacity.

In vitro stimulation of intact rat posterior pituitary by either veratridine or K+ depolarization results in the concomitant release of neurophysins and in a decrease (70-80%) in their carboxyl methylation as measured either with L-[methyl-3H]methionine in the intact lobes after stimulation or in their homogenates with [methyl-3H]S-adenosyl-L-methionine and purified protein carboxyl methyltransferase. A similar reduction in neurophysin methylation (60%) was observed when the arrival of newly synthesized neurophysins at the posterior pituitary was blocked by colchicine. Experimental data indicate that the reduction in neurophysin content of the lobes after 12 h of colchicine treatment (less than 7%) or after in vitro stimulation (about 10%) cannot account for the marked reduction in neurophysin methylation. The results suggest that the granule pool characterized by rapid turnover of neurophysins probably represents the major source of methyl acceptor proteins in the lobe. In spite of the marked reduction in neurophysin methyl accepting capacity observed after stimulation, there was no parallel increase in methyl accepting capacity of the released neurophysins. We propose that a neurophysin subfraction that might be associated with the membrane of releasable granules participates in the methylation reaction in situ.

Animals↗
PubMed1986

Secretion of vasopressin, oxytocin, and two neurophysins from rat hypothalamo-neurohypophyseal explants in organ culture.

Secretion of the neurohypophyseal principles arginine vasopressin, oxytocin, vasopressin-neurophysin, and oxytocin-neurophysin by hypothalamic-neurohypophyseal explants in organ culture was studied. The viability of explant cultures was tested on day 6 by their ability to incorporate 3H-amino acid into acid-precipitable protein, specifically vasopressin-neurophysin. The explants released all four neurosecretory products in a controlled manner during 8 successive days of culture. In addition, these substances were all present in neural lobes and hypothalami at the end of the culture period. Although the tissue retained a 1:1 molar ratio between the hormone and corresponding neurophysin, the levels of neurophysins in culture media were much higher than the levels of hormones. Through immunoprecipitation techniques we determined that both arginine vasopressin and oxytocin were unstable in culture media containing explants or in medium containing 10% heat-inactivated fetal calf serum. This suggests that neurophysins are more stable indicators of secretion by explants than are the corresponding hormones. An increased release of arginine vasopressin, oxytocin, vasopressin-neurophysin, and oxytocin-neurophysin occurred in response to elevated K+ and to increased sodium ions in the medium, and these four substances were released in different molecular proportions. The results indicate that explant cultures may be useful for comparisons of secretion of the two neurophysins and, to a more limited extent, of the two hormones in response to such stimuli.

Animals↗
PubMed1973

Cerebrospinal fluid and ependymal neurophysin.

Neurophysins are "carrier proteins" associated with vasopressin and oxytocin in the neurohypophyseal system. The release of these hormone associated proteins may serve as an indicator of posterior pituitary function. This report describes the measurement of neurophysin in human and monkey plasma and cerebrospinal fluid (CSF) by radioimmunoassay. Tissue neurophysin is also localized in monkey brain by the immunoperoxidase technique. CSF from 68 patients and five monkeys had easily measurable neurophysin in every sample. The concentration of neurophysin in CSF and in plasma of man is 5.4+/-0.30 ng/ml (mean and SEM) and 0.69+/-0.04, respectively. The two means were significantly different (P < 0.001). In paired plasma and CSF specimens which were obtained simultaneously from each of 13 human and five monkey donors, the concentrations of neurophysin in CSF were greater than those of plasma in every case (paired t test, P < 0.001). Neurophysin administered intravenously to dogs did not enter CSF. Using the immunoperoxidase technique, we found neurophysin not only in the supraoptic and paraventricular nuclei, their tracts, and the posterior pituitary, but also in the specialized ependymal tanycytes of the infundibular recess of the third ventricle and in the external layer of the median eminence where capillaries drain into hypophyseal portal vessels. Neurophysin may pass from CSF to portal vessels via tanycytes in a manner similar to that postulated for releasing factors.

Animals↗
PubMed1986

Localization of oxytocin and neurophysin in baboon (Papio anubis) corpus luteum by immunocytochemistry.

Immunoreactive oxytocin is detectable in the corpora lutea of women and cynomolgus monkeys by radioimmunoassay. To localize the presence of oxytocin and neurophysin I in ovarian tissues of subhuman primates, three corpora lutea and ovarian stromal tissues and two Fallopian tubes obtained during the menstrual cycle of the baboon and decidua from two pregnant baboons were examined using highly specific antisera against either oxytocin or neurophysin I and peroxidase-antiperoxidase light microscopy immunohistochemistry. Oxytocin-like as well as neurophysin I-like immunoreactivities were found in some cells of all the corpora lutea only, but could not be demonstrated in ovarian stromal tissues, Fallopian tubes and decidua. Specificity of the immunocytochemical reaction was further confirmed by immunoabsorption of the antiserum with excess oxytocin or neurophysin, after which the immunoreactivities for both oxytocin and neurophysin in the luteal tissue were negative. Similar controls using normal rabbit serum gave no positive staining for either oxytocin or neurophysin. Counterstaining of the positive immunoreactivities for oxytocin and neurophysin I with Mayer's haematoxylin and eosin demonstrated clearly that the oxytocin and neurophysin I appeared as granular material mainly within the cytoplasm of the luteal cells. The localization of immunoreactive oxytocin and neurophysin I in the corpus luteum of the baboon demonstrates directly the presence of these two neurohypophysial peptides within primate luteal cells and suggests their local production.

Animals↗
PubMed1988

Effect of neurophysin on enzymatic maturation of oxytocin from its precursor.

We examined the extent to which rates of enzymatic conversion of the oxytocin biosynthetic precursor to mature peptide are modulated by intramolecular and intermolecular assembly of precursor and polypeptide intermediates. The biosynthesized precursor contains hormone and neurophysin sequences linked by a Gly-Lys-Arg sequence and undergoes enzymatic processing reactions which include endoproteolytic cleavage at the Lys-Arg dibasic sequence, carboxypeptidase B-like exoproteolytic cleavage, and enzymatic amidation. We evaluated the effect of neurophysin on such processing reactions using semisynthetic precursors of oxytocin/bovine neurophysin I and synthetic oxytocinyl precursor intermediates as substrates. Neurophysin I at high concentration (0.7 mM) reduced the rates of carboxy-peptidase B-like conversion of oxytocinyl-Gly-Lys-Arg to oxytocinyl-Gly and the enzymatic amidation of oxytocinyl-Gly to mature (C-terminal amidated) oxytocin. The dependence of rate suppression on the concentrations of peptide substrate and neurophysin I suggested that suppression is due to intermolecular formation of hormone-neurophysin complexes which are aggregated at least to dimers. An analogous intramolecular neurophysin effect was found for endoproteolytic processing of semisynthetic precursors. Endoproteinase Lys-C cleaved the Lys11-Arg12 peptide bond in a native-like semisynthetic precursor at a significantly slower rate than it did an assembly-deficient precursor analogue. The difference in semisynthetic precursor endoproteolysis rates is most substantial at the high concentrations at which the native-like precursor would form dimers but the assembly-deficient analogue would not. The native-like semisynthetic precursor was more stable than the assembly-deficient precursor analogue to tryptic digestion. The concentration-dependent effects of neurophysin, both intramolecularly as a precursor domain and intermolecularly as an interacting protein, are likely to occur in the secretory granules in which the biosynthetic precursors are packaged. The molecular organization of both hormone/neurophysin precursors and the noncovalently complexed hormone-neurophysin intermediates can be expected to play a role in modulating enzymatic processing reactions that lead to mature neurohypophysial hormones.

Animals↗
PubMed1985

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.

Amino Acid Sequence↗