High-performance liquid chromatographic mapping and structural characterization of neurophysin isoforms

Both ion-exchange and reverse-phase HPLC protocols for micromapping of neurophysins have been examined and the structural relationships among the major isoforms identified in the maps have been characterized. Reverse-phase HPLC was found to be especially useful for obtaining fingerprints of the isoforms within each of the two major families of neurophysins, I (oxytocin-related) and II (vasopressin-related), for both bovine and human neurophysins from posterior pituitary sources. From fractionation of the bovine proteins on octylsilyl columns, at least four neurophysins I were identified, one of which corresponds to the intact sequence of 93 residues and three of which vary from the parent by various degrees of carboxyl-terminal truncation. For bovine neurophysin II, two isoforms were identified in the reverse-phase HPLC maps, both of 95 residues, which vary from one another by the residue, either Ile or Val, at position 89. Isoforms were also detected for human neurophysins, including a carboxyl-terminal truncated form of human neurophysin II. All of the major neurophysin isoforms and several of the minor forms were shown to be functionally active as expressed by their binding to peptide ligand affinity matrices. Reverse-phase HPLC mapping on the octylsilyl matrix allowed neurophysin fingerprinting of crude tissue extracts by providing a narrow "window" within which the neurophysins elute but many other polypeptides expected to be present are excluded. The reverse phase HPLC method provides a useful way to obtain isolated neurophysin isoforms for physicochemical characterizations now usually carried out with mixtures of isoforms obtained by ion-exchange chromatography. The method also has characteristics amenable both for high-sensitivity fingerprinting of neurophysin isoforms, from different species and anatomical sources, and as a prelude to microstructural and -functional characterization of the isoforms so isolated.     

Use of reverse-phase high-performance liquid chromatography in structural studies of neurophysins,photolabeled derivatives,and biosynthetic precursors

The neurophysins are a class of hypothalamo-neurohypophyseal proteins that function as carriers of the neuropeptide hormones oxytocin and vasopressin. Currently, we are using reverse-phase high-performance liquid chromatography for structural characterization of the neurophysins, their chemically modified derivatives, and biosynthetic precursors. A cyanopropylsilyl (Zorbax CN) matrix has been found to be efficient and convenient for separation of major tryptic peptides of performic acid, oxidized or reduced, and alkylated neurophysins. Using this peptide mapping system we have studied the site of modification of a photoaffinitylabeled derivative of bovine neurophysin II by separation and identification of covalently modified peptides. In addition, this system has been used for mapping subfemtomole amounts of radioactively labeled biosynthetic precursors of the neurophysins. This procedure has allowed identification of neurophysin sequences within both pre-pro-neurophysins produced by in vitro translation and rat pro-neurophysins produced by in vivo pulse labeling.     

Mapping and isolation of large peptide fragments from bovine neurophysins and biosynthetic neurophysin-containing species by high-performance liquid chromatography

A peptide mapping procedure suitable for rapid analysis and peptide recovery was devised for the neurophysins. Tryptic fragments of performic acid-oxidized bovine neurophysins I and II were fractionated by reverse-phase high-performance liquid chromatography using γ-cyanopropyl-bonded columns. Elutions were achieved using a gradient from triethylammonium phosphate buffer to mixtures of this buffer with increasing proportions of acetonitrile. All tryptic fragments, except for dipeptides, were separated. Assignments of eluted peaks to particular authentic neurophysin peptides were achieved by collection of peaks and determination of their amino acid compositions. The use of this peptide mapping procedure to detect subpicomole amounts of neurophysin peptides in cell-free biosynthetic products was demonstrated.     

The effects of ligands on enzymic carboxyl-methylation of neurophysins

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

Preparative and analytical affinity chromatography of neurophysins on methionyl-tyrosyl-phenylalanyl-aminohexyl-agarose

Methionyl-tyrosyl-phenylalanyl-ω-aminohexyl-agarose was synthesized and shown to be suitable for both the affinity chromatographic purification of neurophysins and the measurement of the ligand binding parameters of these proteins by quantitative affinity chromatography. Bovine neurophysin I binds to the tripeptidyl matrix in 0.4 m ammonium acetate, pH 5.7, conditions under which no binding occurs with the parent ω-aminohexyl-agarose. Subsequent elution can be effected with 0.2 m acetic acid. The affinity matrices obtained have capacities for neurophysin of up to 4 mg/ml gel bed volume and therein provide for the convenient purification of the neurophysins by a two-step buffer-acid elution. [Carbamoyl-¹⁴C]neurophysin I also binds to the ligand-agarose matrix. Using this labeled protein, competitive elution analysis was performed by one-step elution of zones of protein with the binding buffer in the presence of varying amounts of soluble competitive ligand, lysine vasopressin. The characteristic decrease of elution volume of labeled protein with increasing soluble, competing ligand concentration indicates that the affinity matrix interacts biospecifically with neurophysin. This analysis allows the binding affinities for both soluble vasopressin and immobilized tripeptide ligand to be quantitated.     

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     

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.     

Binding studies of polypeptide hormones to bovine neurophysins.

Experimental binding isotherms of [9-glycinamide-1-(14)C]oxytocin and [9-glycinamide-1-(14)C]arginine vasopressin to purified neurophysins I and II at pH = 4.4, 5.4, 6.5, 7.4, and 8.5 and 6 degrees, 22 degrees, and 37 degrees in aqueous buffers are reported. For purposes of comparison, binding isotherms for [4-glycine-1-(14)C]oxytocin to neurophysin II and I in aqueous buffer, and [9-glycinamide-1-(14)C]oxytocin to neurophysin II in dimethylsulfoxide under selected conditions are also reported. A brief discussion of the interpretation of binding isotherms is entered into and apparent binding constants are derived. The results indicate that the interpretations presented in the literature up to now are much too simple. There are, in contrast, multiple binding sites of oxytocin and vasopressin to the neurophysins and large temperature dependences of the number of sites and their binding constants. We find, in fact, that at 37 degrees the binding of neurohypophysial hormones to the supposed storage proteins is rather weak even at the pH of maximum binding.     

Presence of neurophysins I and II in the human pineal gland: Comparison with the content of neurohypophyseal hormones

We have previously measured the individual content of immunoreactive vasopressin (AVP), oxytocin (OT) and vasotocin (AVT) in 155 human pineal glands, and report here identification and measurement of the neurophysin (Np) content of the same glands, using specific homologous human neurophysin I (HNp I) and neurophysin II (HNp II) radioimmunoassays. Median values for HNp I were for men 47 ng/gland (range, 5 to 1360) and for women 24 ng/gland (range, 5 to 1000); median values for HNp II were respectively 7 ng/gland (range, 2 to 191) and 15 ng/gland (range, 2 to 356) with no significant difference between men and women for HNp I and HNp II but a significant difference (p<0.001) between HNp I and HNp II for both sexes. Gel filtration showed that pineal neurophysins were eluted at the same volume as both standard Np and Np from human posthypophyses used as controls. HNp I correlated both with AVP (r_s=0.54 for men and 0.55 for women) and OT (r_s=0.86 for men and 0.57 for women) but not with AVT, while HNp II correlated with AVP (r_s=0.52 for men and 0.53 for women) and OT (r_s=0.92 for men and 0.50 for women) but not with AVT. This study thus confirms the presence of two neurophysins in the human pineal gland and further indicates that they are related to AVP and OT concentrations in the same gland. The results also imply, however, that the presence of immunoreactive AVT (more probably a closely related peptide) is independent of the neurophysins.     

Neurophysin heterogeneity. Difference between newly formed and aged neurosecretory granules

Neurosecretory granules from bovine neurohypophyses were isolated on iso-osmotic gradients. The content of the granules was analyzed by analytical and two-dimensional gel electrophoreses. The distributions in the gels of vasopressin precursor and neurophysins were detected by radioimmunoassays. Analytical gel electrophoresis of the content of a crude granule preparation showed the presence of different populations of neurophysin molecules. Further analysis demonstrated that vasopressin-neurophysin and oxytocin-neurophysin can be subdivided into molecules with different pI values. Whereas newly formed granules showed two main spots of neurophysin with pI of 5.0 and 5.6, aged granules contain in addition to these different populations of neurophysin-like material, some of which had a basic pI. Vasopressin precursor activity was detected in spots containing proteins with acidic pI and Mr approximately 18,000 and also in proteins of Mr = 8,000-10,000 migrating in the basic region of the gel. The results suggest that in the neural lobe there is an aging process which gives rise to several subpopulations of neurophysins. The different forms of vasopressin-associated bovine neurophysin and oxytocin-associated bovine neurophysin are only found in the granules which are not required for release.     

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.     

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.     

Secretion of neurophysins during luteal regression in the goat

In non-pregnant goats, ovariectomy on day 12 of oestrous cycle resulted in parallel decrease of oxytocin and progesterone jugular concentration. Similarly, luteolysis, indicated by decreasing progesterone concentration, was accompanied by simultaneous release of oxytocin and oxytocin-associated neurophysins (mean of neurophysin I and II). It is suggested that the neurophysins are secreted concomitantly with oxytocin by the ovary during luteal regressions in the goat.     

Preparation of Porcine Neurophysin Proteins by High Performance Liquid Chromatography

Abstract: The crude neurophysin containing extract from posterior lobes of porcine pituitaries was roughly purified by gel chromatography. 15 mg of the lyophilized neurophysin complex were completely separated by HPLC yielding in neurophysin I₁ (3.6 mg), I₂ (4.0 mg), II (4.6 mg) and III (1.9 mg). All of the neurophysins were homogenous by PAGE and SDS-electrophoresis, isoelectrofocussing, amino-acid composition and N- and C-terminal amino acid analysis. In conclusion, HPLC is a reliable and quick method for the preparation of pure neurophysins.     

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     

Immunoreactive material resembling vertebrate neuropeptides and neurophysins in the brain,suboesophageal ganglion,corpus cardiacum and corpus allatum of the dictyopteran Periplaneta americana L.

Summary The peroxidase-antiperoxidase technique (Vandesande and Dierickx 1976) with antibodies raised against several vertebrate neuropeptides and neurophysins, was applied] to 4-m Paraplast-embedded serial sections of in situ fixed brains and adjacent suboesophageal ganglion (SOG), corpora cardiaca (CC) and corpora allata (CA) of the blattarian insect Periplaneta americana L. Substances immunologically related to bovine neurophysin I (NPI) and II (NPII), synthetic arginine vasopressin (AVP) and synthetic oxytocin (OT) were found to be differentially distributed in the central nervous system. The differences among all four antigens demonstrated became clearly evident by immunohistochemical double-staining procedures (Vandesande 1983); no overlapping was observed. The same double-staining technique revealed that these vertebrate-type substances occur in other neurosecretory cells and axons than those containing CRF- and ACTH-like material as reported earlier (Verhaert et al. 1984).     

Properties of purified folate-binding proteins from chronic myelogenous leukemia cells

Folate-binding protein(s) from chronic myelogenous leukemia cells have been purified using acid dialysis, ammonium sulfate fractionation and affinity chromatography. The purified preparation which migrates as a single band on disc electrophoresis could be separated by DEAE agarose chromatography into two folate-binding proteins (binders I and II) which bind molar equivalents of folic acid. One binder (I) eluted from DEAE at 1 mM sodium phosphate, pH 6.0, and the other (II) at 100 mM sodium phosphate, pH 7.4. Analysis of the purified mixture, which contained more than 90% binder II, by sedimentation equilibrium centrifugation indicated a homogeneous protein with a calculated molecular weight of 44000. Antiserum raised against the purified mixture gave a single precipitin line by immunodiffusion against a preparation of partially purified cell lysate.Hydrolysis of the more acidic binder (II) with neuraminidase converted it to a weakly acidic protein similar to binder I suggesting that these binders are glycoproteins which differ in sialic acid content. With isoelectric focusing, the binding of folic acid would be demonstrated at pH 6.7, 7.3, 7.8 and 8.2 for binder I, and at pH 5.1, 5.8 and 6.5 for binder II. Binders I and II had equally high affinity for folic acid and dihydroflate, lower affinity of N⁵-methyl-tetrahydrofolate, and no apparent affinity for N⁵-formytetrahydrofolate or methotrexate.     

Isolation,purification, and properties of mammalian and avian liver and yeast fatty acid synthetase acyl carrier proteins

Acyl carrier proteins were isolated from rat, human, pigeon, and chicken liver and yeast fatty acid synthetase complexes. These proteins were separated from the other proteins of subunit I of each complex by ultrafiltration after dialysis of subunit I for 3 h against low ionic strength buffer [Qureshi et al. (1974) Biochem. Biophys. Res. Commun.60, 158–165]. Subunit I of each fatty acid synthetase was previously separated from subunit II by affinity chromatography on Sepharose ?-aminocaproyl pantetheine and subsequent sucrose density gradient centrifugation. The separated acyl carrier proteins were then subjected to gel filtration on a Sephadex G-50 column. The proteins obtained from each fatty acid synthetase were homogeneous with respect to size and charge on gel filtration, paper and disc gel electrophoresis, and chromatography on diethylaminoethyl-cellulose. The physical properties and the ability to accept acetyl and malonyl groups from acetyl- and malonyl-CoA in the presence of transacylase were similar to those of Escherichia coli acyl carrier protein. These proteins ranged in molecular weight from 7500 to 10,000. Each of the acyl carrier proteins showed the presence of β-alanine and each yielded acetyl- and malonyl-A₁ and A₂ peptic peptides, thus indicating the presence of a 4′-phosphopantetheine prosthetic group in each. They differed somewhat from each other in amino acid composition, but each had a high number of negatively charged (aspartate and glutamate) amino acid residues.     

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.     

Effect of phosphate on the macromolecular state of bovine neurophysin

Bovine neurophysin II has been subjected to equilibrium sedimentation in 0.1 m phosphate, pH 5.8, and in 0.1 I acetate, pH 5.6. Under the former conditions the molecular weight is 20,000, which is consistent with the concept that neurophysin is dimeric in the phosphate medium. In acetate the molecular weight varies with neurophysin concentration in conformity with this carrier protein being a monomer-dimer system governed by an association equilibrium constant of 5800 m^?1. This investigation has thus confirmed that the disparity between two previous ultracentrifuge studies of bovine neurophysin II reflected a genuine difference in the macromolecular state of the protein under the conditions employed. The implications of this difference are discussed in relation to the nature of the macromolecular interactions that are responsible for the cooperative binding of oxytocin to neurophysin.