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At least 19 recordsLinked to original sources
PubMed1979

Identification and observation of alkyl proton resonances of the amino-terminal residues of bovine neurophysins. Evidence for conformational differences between neurophysin-I and neurophysin-II.

Analysis of the 220 MHz proton magnetic resonance spectra of bovine neurophysins-I and -II and of the effects of pH and succinylation of these spectra has allowed identification of the -CH3 proton resonances of the amino-terminal alanine of both proteins and of the -CH3 resonances of methionine-2 of neurophysin-II. The alanine -CH3 resonance of neurophysin-I is a sharp doublet at all pH values between 1 and 10.5 indicating relatively few restrictions on its mobility. By contrast, the -CH3 resonances of the amino-terminal alanine and methionine-2 of neurophysin-II undergo pH-dependent changes in broadening compatible with the formation of an intramolecular salt-bridge at neutral pH between the protonated alpha-amino and an unprotonated side chain carboxyl. The results suggest that differeces in the properties of the two proteins are partially mediated by conformational differences involving their amino-terminal sequences. The potential usefulness of the amino-terminal resonances as n.m.r. 'reporter' signals is additionally demonstrated by studies of the effects of spin labels on the neurophysin-I amino-terminal alanine resonance; these studies place the amino-terminus of neurophysin-I approximately 14 A from residue 3 of peptides bound to the strong neurophysin hormone-binding site.

Amino Acids↗
PubMed1976

Immunocytochemical study of the hypothalamo-neurohypophysial system. I. Localization of neurosecretory neurons containing neurophysin-I and neurophysin-II in the domestic pig.

Antibodies raised against porcine neurophysin-I and porcine neurophysin-II using an injection regime in rabbits over a short time period, were used to localize neurophysin-I and neurophysin-II in hypothalamic neurosecretory elements of the domestic pig. In transverse section, neurophysin-II containing cells were more abundant in the dorsal medial region of the rostral supraoptic nucleus (SON) as compared with the distribution of neurophysin-I neurons. The main bulk of the cells of the SON were heavily stained for neurophysin-I with neurophysin-II containing cells positioned dorsal from the edge of the optic chiasma. Neurosecretory cells of the SON as seen in sagittal section also showed a differential staining for neurophysins-I and -II. Rostral regions of the pig paraventricular nucleus (PVN) contained magnocellular elements near the third ventricle which were stained predominantly for neurophysin-II. In regions corresponding to the caudal PVN there appeared two populations of neurosecretory neurons: (a) an area of cells adjacent to the third ventricle which contained neurophysin-II antigen and (b) a group of densely populated cells in the dorsal-lateral region which was stained for neurophysin-I. The results support the existence in the pig of at least two distinct populations of neurosecretory neurons corresponding to the neurophysin-I and neurophysin-II neurosecretory system.

Animals↗
PubMed1983

Identification of human neurophysins: complete amino acid sequences of MSEL- and VLDV-neurophysins.

Two human neurophysins have been purified from acetone-desiccated posterior pituitaries by acidic extraction, molecular sieving, and ion-exchange chromatography. The complete amino acid sequence of each protein has been determined by using a sequencer and characterizing two sets of overlapping enzymic peptides. The two neurophysins belong to two structural families previously defined as MSEL- and VLDV-neurophysins according to the nature of the residues in positions 2, 3, 6, and 7. (MSEL-neurophysins contain methionine-2, serine-3, glutamic acid-6, and leucine-7; VLDV-neurophysins contain valine-2, leucine-3, aspartic acid-6, and valine-7.) Human MSEL-neurophysin has only 93 residues instead of 95 usually found in MSEL-neurophysins from other mammalian species, probably because of a deletion of amino acids 91 and 92. Compared with bovine MSEL-neurophysin, nine variations (seven substitutions and two deletions) are observed. Human VLDV-neurophysin has 93 residues, as do the other mammalian VLDV-neurophysins. There are 11 substitutions when the comparison is made with bovine VLDV-neurophysin. Between the two human neurophysins, there are 26 variations. However, the central parts of the proteins (residues 10-70) are nearly identical. Furthermore, in this region identical substitutions are found in positions 29 and 60 of both neurophysins, suggesting either a single exon or some relationship between the two corresponding genes.

Amino Acid Sequence↗
PubMed1989

Ostrich MSEL-neurophysin belongs to the class of two-domain "big" neurophysin as indicated by complete amino acid sequence of the neurophysin/copeptin.

Mammalian neurohypophyseal hormones, oxytocin and vasopressin, are known to be synthesized as part of two larger precursors containing, respectively, a VLDV-neurophysin and a MSEL-neurophysin together with its associated glycopeptide. Starting from ostrich neurohypophyses, a "big" neurophysin was isolated and chemically characterized. Following sequence determination of the CNBr-derived fragments and of peptides obtained from trypsin and V8-protease digestion of the oxidized protein, this "big" neurophysin was found to contain an MSEL-neurophysin moiety (94 residues) still covalently associated with the COOH-terminal glycopeptide (38 residues, copeptin). This study demonstrates that the ostrich MSEL-neurophysin sequence closely resembles all known MSEL-neurophysin sequences and that, furthermore, it does not contain the single amino acid insertion shown previously in the ostrich VLDV-neurophysin. It is also shown that the stretch of amino acids, linking the MSEL-neurophysin and the copeptin, is clearly different from its mammalian homologues and lacks the Arg residue normally recognized by the cleaving enzyme. This study also demonstrates that the ostrich copeptin is more closely related to the amphibian copeptin sequence than to its mammalian homologue, leading to the hypothesis that two families of copeptin molecules might exist. Thus, the ostrich MSEL-neurophysin-copeptin molecule is the first "big" neurophysin reported in birds and, together with the guinea pig and amphibian homologues, represents the third example of partial or no neurophysin-copeptin cleavage.

Amino Acid Sequence↗
PubMed1973

Intra-axonal transport and turnover of neurophysins in the rat. A proposal for a possible origin of the minor neurophysin component.

1. Radioactivity associated with the three neurophysins in the neural lobe of the rat was determined at intervals up to 5 weeks after an intracisternal injection of [(35)S]cysteine. 2. The radioactivity associated with the two major neurophysins (one supposedly associated with vasopressin and the other with oxytocin) increased linearly for 12h after the injection and the ratio of the rates of increase in the two proteins was very similar to the ratio of vasopressin to oxytocin in the gland. 3. From 12h onwards the radioactivity associated with each major neurophysin declined exponentially but the half-life of the supposed oxytocin-neurophysin (13.3 days) was shorter than that for the supposed vasopressin-neurophysin (19.8 days). 4. The kinetics of labelling of the minor neurophysin was quite different from that of the two major ones. It became slowly labelled during 3-5 days after injection and the radioactivity hardly decreased during the following 4 weeks. 5. The data could support the hypothesis that the minor neurophysin is a metabolic product of oxytocin-neurophysin. The exponential rate of disappearance of radioactivity from oxytocin-neurophysin and the minor component taken together has a rate constant similar to that for vasopressin-neurophysin (e.g. half-life=18.9 days).

Animals↗
PubMed1987

Guinea pig MSEL-neurophysin. Sequence comparison of eight mammalian MSEL-neurophysins.

The amino acid sequence of guinea pig MSEL-neurophysin has been determined using tryptic peptides derived from the performic acid-oxidized protein and staphylococcal proteinase peptides obtained from the reduced-carboxamidomethylated neurophysin. Guinea pig MSEL-neurophysin consists of a 93-residue polypeptide chain that shows 12 substitutions and 2 deletions when compared to bovine MSEL-neurophysin. It displays the highest number of variations among known mammalian MSEL-neurophysins. These variations are mainly found in the C-terminal region (residues 88-93). Moreover guinea pig MSEL-neurophysin, like rat homologous protein, exhibits substitutions in positions 2, 5, 29 and 81 and lacks an arginine in the penultimate position. Comparison between eight mammalian MSEL-neurophysins reveals a highly conserved region (residues 1 to 88) and a hypervariable region (residues 89 to 93/95). On the other hand the eight species examined are endowed with arginine vasopressin except pig, which has a lysine vasopressin. In the vasopressin-MSEL-neurophysin precursor, the hormonal moiety and the MSEL region of neurophysin (residues 1-9) are encoded by a common exon in ox, rat and man; it can be concluded that this exon is evolutionarily conservative in contrast to the one encoding the C-terminal region of MSEL-neurophysin.

Amino Acid Sequence↗
PubMed1981

Nature of the immunoreactive neurophysins in ectopic vasopressin-producing oat cell carcinomas of the lung. Demonstration of a putative common precursor to vasopressin and neurophysin.

In an attempt to delineate the nature of the immunoreactive neurophysins in oat cell carcinomas of the lung with ectopic vasopressin production, tumor neurophysins were characterized by gel filtration and by electrophoresis. In all of the five tumor tissues, activities of both vasopressin and nicotine-stimulated neurophysin (NSN) determined by radioimmunoassay were demonstrated. A small amount of oxytocin as well as estrogen-stimulated neurophysin was detected in three of the tissues. When tissue extract was subjected to Sephadex G-50 gel filtration in 0.2 N acetic acid, the major portion of immunoreactive NSN emerged in the fractions corresponding to the molecular size of 10,000. The migration pattern of NSN in these fractions on electrophoresis was qualitatively the same as that of NSN extracted from human posterior pituitary glands. In addition to this major neurophysin, immunoreactive NSN with the molecular size of 20,000 was consistently demonstrated in three tumor extracts. This high molecular weight form of neurophysin represented 6.5--8.7% of total NSN immunoactivities in each tumor extract and its elution profile was not changed when analyzed under denaturating conditions in 6 M guanidine hydrochloride. On electrophoresis, it migrated near the gamma globulin region; however, the peak was broad suggesting that it consists of more than two different molecular populations. A substantial portion of the high molecular weight NSN appears to be a glycoprotein judging from its binding to concanavalin A. When the high molecular weight from of neurophysin was incubated with trypsin, essentially all of the activities were converted into NSN with the molecular size of 10,000. Moreover, an equimolar amount of vasopressin was liberated after the treatment, the elution pattern of which closely resembled that of synthetic arginine vasopressin. When a lower concentration of trypsin was used, some of the 20,000-dalton neurophysin exhibited activities of both NSN and vasopressin. Since the antivasopressin serum used in this study appeared to be directed toward the ring portion side of vasopressin, these results suggest that this 20,000-dalton neurophysin is, in all probability, a common precursor to vasopressin and neurophysin, and that vasopressin may be located in the middle of the precursor molecule.

Aged↗
PubMed1975

Identification of the vasopressin-neurophysin II and the oxytocin-neurophysin I producing neurons in the bovine hypothalamus.

Immuno-enzyme histochemical investigations on the bovine hypothalamus showed that vasopressin and oxytocin are synthetized in separate neurons. Moreover, it was found that the vasopressin-producing neurons are identical with the neruophysin II-producing neurons, while the oxtocin producing neurons correspond with the neurophysin I-producing neurons. From this result it is concluded that, in the species studied, neurophysin I is the carrier protein of oxytocin and that neurophysin II is the carrier protein of vasopressin. The bovine vasopressin-neurophysin II producing neurons and the oxytocin-neurophysin I producing neurons are both present as well in the suparoptic nuclei as in the paraventricular nuclei. In the supraoptic nuclei, the vasopressin-neurophysin II neurons predominate in number; in the paraventricular nuclei, the oxytocin-neurophysin II neurons predominate. The two kinds of bovine neurosecretory neurons are partly localized in separate areas. Moreover, the vasopressin-neurophysin II neurons and the oxytocin-neurophysin I neurons show distinct morphological differences.

Animals↗
PubMed1979

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

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 wiht 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.

Animals↗
PubMed1982

Partial digestion of neurophysins with proteolytic enzymes: unusual interactions between bovine neurophysin II and chymotrypsin.

Bovine neurophysin II was partially digested by chymotrypsin and by chymotrypsin followed by carboxy-peptidase B to produce large fragments collectively representing deletions of residues 1-5 and 91-95. All such fragments were capable of binding peptides to the principal hormone-binding site of neurophysin with normal or near-normal affinity, indicating that residues 1-5 and 91-95 do not directly participate in binding. In addition, preliminary results with thermolysin-derived fragments suggested that residue 6 does not participate in peptide binding. During the course of chymotrypsin studies, it was demonstrated that bovine neurophysin II behaves as a transient competitive inhibitor of chymotrypsin; for neurophysin-peptide complexes, Ki congruent to 8 x 10(-6) M. This inhibition is dependent on neurophysin conformation and is relieved by the anomalous preferential splitting by chymotrypsin of Arg-Arg and Phe-Pro bonds near the carboxyl terminus of neurophysin II. It is suggested that this phenomenon might reflect the interaction of neurophysin II with a chymotrypsin-related enzyme in the pituitary. One approach used in the study of binding properties of proteolytically modified neurophysin was affinity chromatography; the preparation and properties of a conveniently prepared affinity column for neurophysin are described.

Chromatography, Affinity↗
PubMed2007

Contributions of the interdomain loop, amino terminus, and subunit interface to the ligand-facilitated dimerization of neurophysin: crystal structures and mutation studies of bovine neurophysin-I.

Current evidence indicates that the ligand-facilitated dimerization of neurophysin is mediated in part by dimerization-induced changes at the hormone binding site of the unliganded state that increase ligand affinity. To elucidate other contributory factors, we investigated the potential role of neurophysin's short interdomain loop (residues 55-59), particularly the effects of loop residue mutation and of deleting amino-terminal residues 1-6, which interact with the loop and adjacent residues 53-54. The neurophysin studied was bovine neurophysin-I, necessitating determination of the crystal structures of des 1-6 bovine neurophysin-I in unliganded and liganded dimeric states, as well as the structure of its liganded Q58V mutant, in which peptide was bound with unexpectedly increased affinity. Increases in dimerization constant associated with selected loop residue mutations and with deletion of residues 1-6, together with structural data, provided evidence that dimerization of unliganded neurophysin-I is constrained by hydrogen bonding of the side chains of Gln58, Ser56, and Gln55 and by amino terminus interactions, loss or alteration of these hydrogen bonds, and probable loss of amino terminus interactions, contributing to the increased dimerization of the liganded state. An additional intersubunit hydrogen bond from residue 81, present only in the liganded state, was demonstrated as the largest single effect of ligand binding directly on the subunit interface. Comparison of bovine neurophysins I and II indicates broadly similar mechanisms for both, with the exception in neurophysin II of the absence of Gln55 side chain hydrogen bonds in the unliganded state and a more firmly established loss of amino terminus interactions in the liganded state. Evidence is presented that loop status modulates dimerization via long-range effects on neurophysin conformation involving neighboring Phe22 as a key intermediary.

Allosteric Regulation↗
PubMed1990

Identification of two types of neurophysins in Xenopus laevis neurointermediate pituitary homologous to mammalian MSEL- and VLDV-neurophysins.

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).

Amino Acid Sequence↗
PubMed1999

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.

Animals↗
PubMed1990

Non-mammalian "big" neurophysins--complete amino acid sequence of a two-domain MSEL-neurophysin from goose.

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.

Amino Acid Sequence↗
PubMed1994

Bony fish neurophysins. Identification of MSEL- and VLDV-neurophysins of the pollack (Pollachius virens).

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.

Amino Acid Sequence↗