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

Abstract

NMR was used to monitor the binding to neurophysin of oxytocin and 8-arginine-vasopressin, /sup 15/N labeling being used to identify specific backbone /sup 15/N and /sup 1/H 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 for 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 neutrophysin chain. The behavior of both oxytocin and vasopressin on binding was consistent withmore » 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 /sup 15/N resonances in the bound state at low pH occurred without a corresponding change in the resonances of equilibrating free hormone. The results suggest significant conformational alteration in neurophysin-hormone complexes at low pH possibly associated with protonation of the carboxyl group of the hormone-protein salt bridge.« less

Authors:
;
Publication Date:
Research Org.:
Rockefeller Univ., New York, NY
OSTI Identifier:
5400501
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; (United States); Journal Volume: 26:20
Country of Publication:
United States
Language:
English
Subject:
62 RADIOLOGY AND NUCLEAR MEDICINE; OXYTOCIN; CONFIGURATION INTERACTION; NUCLEAR MAGNETIC RESONANCE; PROTEINS; VASOPRESSIN; ARGININE; CHEMICAL SHIFT; NITROGEN 15; NMR SPECTRA; OVERHAUSER EFFECT; PH VALUE; PROTONS; AMINO ACIDS; BARYONS; CARBOXYLIC ACIDS; ELEMENTARY PARTICLES; FERMIONS; HADRONS; HORMONES; ISOTOPES; LIGHT NUCLEI; MAGNETIC RESONANCE; NITROGEN ISOTOPES; NUCLEI; NUCLEONS; ODD-EVEN NUCLEI; ORGANIC ACIDS; ORGANIC COMPOUNDS; PEPTIDE HORMONES; PITUITARY HORMONES; RESONANCE; SPECTRA; STABLE ISOTOPES 550601* -- Medicine-- Unsealed Radionuclides in Diagnostics

Citation Formats

Live, D.H., and Cowburn, D.. Binding of oxytocin and 8-arginine-vasopressin to neurophysin studied by /sup 15/N NMR using magnetization transfer and indirect detection via protons. United States: N. p., 1987. Web.
Live, D.H., & Cowburn, D.. Binding of oxytocin and 8-arginine-vasopressin to neurophysin studied by /sup 15/N NMR using magnetization transfer and indirect detection via protons. United States.
Live, D.H., and Cowburn, D.. 1987. "Binding of oxytocin and 8-arginine-vasopressin to neurophysin studied by /sup 15/N NMR using magnetization transfer and indirect detection via protons". United States. doi:.
@article{osti_5400501,
title = {Binding of oxytocin and 8-arginine-vasopressin to neurophysin studied by /sup 15/N NMR using magnetization transfer and indirect detection via protons},
author = {Live, D.H. and Cowburn, D.},
abstractNote = {NMR was used to monitor the binding to neurophysin of oxytocin and 8-arginine-vasopressin, /sup 15/N labeling being used to identify specific backbone /sup 15/N and /sup 1/H 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 for 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 neutrophysin 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 /sup 15/N resonances in the bound state at low pH occurred without a corresponding change in the resonances of equilibrating free hormone. The results suggest significant conformational alteration in neurophysin-hormone complexes at low pH possibly associated with protonation of the carboxyl group of the hormone-protein salt bridge.},
doi = {},
journal = {Biochemistry; (United States)},
number = ,
volume = 26:20,
place = {United States},
year = 1987,
month =
}
  • Arginine vasopressin (AVP) and oxytocin (OXT) are posterior pituitary hormones. AVP is involved in fluid homeostasis, while OXT is involved in lactation and parturition. AVP is derived from a larger precursor, prepro-arginine-vasopressin-neurophysin II (prepro-AVP-NP II; AVP), and is physically linked to prepro-oxytocin-neurophysin I (prepro-OXT-NPI1; OXT). The genes for AVP and OXT are separated by only 12 kb of DNA in humans, whereas in the mouse 3.5 kb of intergenic sequence lies between Avp and Oxt. Interspecific backcross analysis has now been used to map the Avp/Oxt complex to chromosome 2 in the mouse. This map position confirms and extends themore » known region of linkage conservation between mouse chromosome 2 and human chromosome 20. 16 refs., 2 figs., 1 tab.« less
  • L(/sup 35/S)Cys-arginine vasopressin, -oxytocin, and -somatostatin were purified from hypothalami and neurohypophyses 4 h after rats received L(/sup 35/S)Cys via the third ventricle. After acetic acid extraction, Sephadex G-25 filtration, and chemoadsorption to C18-silica (Sep-Pak cartridges), the labeled peptides were rapidly separated by gradient elution, reversed phase, high pressure liquid chromatography (HPLC). The identity and isotopic purity of the labeled peptides were determined by several reversed phase HPLC procedures in conjunction with chemical modification. The labeled peptide fractions were at least 50% radiochemically pure. Using this HPLC isolation procedure, incorporation of L-(/sup 35/S)Cys into each peptide was determined in hydratedmore » and dehydrated rats. Label incorporation into arginine vasopressin and oxytocin in the hypothalamus and the neurohypophysis of dehydrated rats was 2-3 times greater than that in hydrated rats. Incorporation of label into hypothalamic and neurohypophyseal somatostatin was unaffected by the hydration state of the animal. This procedure thus provides a very rapid, but sensitive, set of techniques for studying the control of small peptide biosynthesis in the brain.« less
  • The resonances of the C/sup ..cap alpha../ and C/sup ..beta../ protons of the cystyl, asparaginyl, and aromatic residues of (8-arginine)vasopressin (AVP) in D/sub 2/O at pD 3.8 and 20/sup 0/C were assigned in a rigorous manner by the use of isotopic isomers of AVP that contain specific replacements of protons by deuterons and by comparison of /sup 1/H NMR characteristics of AVP to those of (8-lysine)vasopressin (LVP) and oxytocin (OT). Although there is extensive overlap of resonances of C/sup ..beta../ protons even at 360 MHz, all of the chemical shifts of these protons and most of the couplings between themmore » and their vicinal C/sup ..cap alpha../ protons could be determined, at least to a first approximation. It was concluded that the cyclic moieties (residues 1-6) of AVP, LVP, and OT possess essentially the same overall backbone conformation, and that the side-chain conformation - or rotamer populations - about the C/sup ..cap alpha../-C/sup ..beta../ bonds of the cystyl residue (positions 1 and 6), the tyrosyl residue (position 2), and the asparaginyl residue (position 5) are similar. This study indicates that selective replacements of C/sup ..beta../ protons by deuterons are necessary to improve the accuracy of coupling constants extracted from 360-MHz spectra of a AVP for use in conformational analysis.« less
  • The arginine vasopressin (AVP) gene was sequenced in a pedigree with familial central diabetes insipidus (DI). When polymerase chain reaction-amplified DNAs from affected subjects were subjected to polyacrylamide gel electrophoresis, fragments including exon 2 displayed two additional, slower migrating bands. These extra bands represented DNA heteroduplexes, indicating that there was a deletion or insertion mutation in exon 2. As the region with such a mutation was identified by direct sequence analysis, polymerase chain reaction-amplified fragments including the region were subcloned and sequenced. A 3-basepair deletion (AGG) out of two consecutive AGG sequences (nucleotides 1824-1829) was identified in one of twomore » alleles. The cosegregation of the mutation with the DI phenotype in the family was confirmed by restriction enzyme analyses. This mutation should yield an abnormal AVP precursor lacking Glu[sup 47] in its neurophysin-II (NP) moiety. Since Glu[sup 47] is essential for NP molecules to form a salt bridge with AVP, it is very likely that the function of NP as a carrier protein for AVP would be impaired. The authors suggest that AVP would undergo accelerated proteolytic degradation, and this mechanism would be involved in the pathogenesis of DI in this pedigree. 34 refs., 4 figs., 2 tabs.« less
  • The effect of cysteamine injection on the in vivo incorporation of (/sup 35/S)cysteine into somatostatin-14 (SRIF-14), SRIF-28, arginine vasopressin (AVP), and oxytocin (OXT) in rat hypothalamus was studied. (/sup 35/S)Cysteine was injected into the third ventricle 1 h, 4 h, or 1 week after cysteamine (300 mg/kg, sc) injection; animals were killed 4 h later. The drug was found to substantially reduce immunoreactive SRIF levels, but not OXT or AVP, 4 h after its injection. Cysteamine also caused large reductions in label incorporation into SRIF-14, SRIF-28, and OXT 1 and 4 h after drug injection. However, (/sup 35/S)cysteine incorporation intomore » AVP was increased substantially at these time points, while that into acid-precipitable protein was normal. One week after cysteamine injection, label incorporation into all hypothalamic peptides was normal. Cysteine specific activity was also measured after (/sup 35/S)cysteine injection and was found to be similar in treatment and control groups. The results suggest that cysteamine inhibits the syntheses of SRIF-14, SRIF-28, and OXT and stimulates that of AVP.« less