Structural study of the N-terminal segment of neuropeptide tyrosine

Forest, Mylène; Martel, Jean Claude; St‐Pierre, Serge; Quirion, Rémi; Fournier, Alain

doi:10.1021/jm00168a014

Cited by 53 publications

(22 citation statements)

References 5 publications

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“…The Boc-amino acids with appropriate side-chain protection were obtained from Richelieu Biotechnologies (St-Hyacinthe, Qudbec). The completed peptide was cleaved from the resin support and deprotected by treatment with HF (29).…”

Section: Methodsmentioning

confidence: 99%

Characterization of melanotropin-release-inhibiting factor (melanostatin) from frog brain: homology with human neuropeptide Y.

Chartrel¹,

Conlon²,

Danger³

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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A polypeptide was purified from frog brain extracts on the basis of its ability to inhibit a-melanotropin release from perifused frog neurointermediate lobes. Based on Edman degradation, amino acid analysis, and peptide mapping, the primary structure of this frog melanotropin-releaseinhibiting factor (melanostatin) was determined to be H-TyrPro-Ser-Lys-Pro-Asp-Asn-Pro-Gly-Glu-Asp-Ala-Pro-Ala-GluAsp-Met-Ala-Lys-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-AsnLeu-Ile-Thr-Arg-Gln-Arg-Tyr-NH2. Frog melanostatin belongs to the pancreatic polypeptide/neuropeptide Y/peptide YY family, and the structure of this peptide differs from that of human neuropeptide Y by only one amino acid substitution in position 19. A synthetic replicate of frog melanostatin is coeluted with the native peptide on HPLC and is highly potent in inhibiting a-melanotropin secretion in vitro (IC50 = 60 nM).Both the anterior lobe and the intermediate lobe of the pituitary are regulated by hypophysiotropic factors originating from the hypothalamus (1, 2). Five families of hypothalamic neuropeptides involved in the control of anterior pituitary hormone secretion have now been characterized, namely, thyrotropin-releasing hormone (3, 4), gonadotropinreleasing hormone (5), somatostatin (6), corticotropinreleasing hormone (7), and growth hormone-releasing hormone (8,9 (20)(21)(22). However, many studies failed to demonstrate that these peptides meet the criteria expected of a physiological melanotropin-release-inhibiting factor (melanostatin) (23)(24)(25). We report here the purification, sequence analysis, and total synthesis of a 36-residue peptide that is highly potent in inhibiting the secretion of a-melanotropin from frog neurointermediate lobe in vitro. MATERIALS AND METHODSPurification Procedure. Adult frogs (Rana ridibunda) were obtained from a commercial source (Coudtard, St-Hilaire de Riez, France). The brains from 1200 animals (94.5 g, wet weight) were collected on dry ice and kept frozen. The tissue was boiled for 15 min in 0.5 M acetic acid and homogenized in a Waring blendor. After centrifugation (10,000 x g for 30 min at 40C), the supernatant was pumped at a flow rate of 2 ml/min through ten Sep-Pak C18 cartridges (Waters Associates) connected in series. Bound material was eluted with 70% (vol/vol) acetonitrile in water and lyophilized. The dry extract was dissolved in 10 ml of 1% (vol/vol) trifluoroacetic acid and chromatographed on a 2.5 x 100-cm column of Sephacryl S-100 (Pharmacia-LKB) equilibrated with 1 M acetic acid at a flow rate of 2 ml/min. Fractions (10 ml) were collected and absorbance was measured at 280 nm (Fig. 1A).The fractions exhibiting melanotropin-release-inhibiting activity were pooled and 50% of the total material was analyzed by HPLC.The active fraction was pumped at a flow rate of 2 ml/min onto a 1 x 25-cm Vydac 218 TP510 C18 column (The Separations Group) equilibrated with 0.1% trifluoroacetic acid and eluted at a flow rate of 2 ml/min, using the gradient indicated in Fig. 1B. Absorbance was measured at 214 and 280 ...

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Section: Methodsmentioning

confidence: 99%

Characterization of melanotropin-release-inhibiting factor (melanostatin) from frog brain: homology with human neuropeptide Y.

Chartrel¹,

Conlon²,

Danger³

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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“…benzotriazol-l-yl-oxy-tris(dimethylamino)-phosphonium hexafluorophosphate (BOP) activation and iV-Boc protection [18]. A combined method was also described for the (1 -35)peptide of human neuropeptide Y using solid-phase synthesis by diisopropylcarbodiimide and 1 -hydroxybenzotriazole (HOBt) activation and W-9-fluorenylmethoxycarbonyl (Fmoc) protection followed by enzymatic attachment of the C-terminal tyrosine amide [ 191.…”

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confidence: 99%

Neuropeptide Y

Mierke

Dürr

Kessler

et al. 1992

European Journal of Biochemistry

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The 36-amino-acid neuropeptide Y (human), which is one of the most potent vasoconstrictors and which exhibits a number of other biological functions, has been synthesized using automated peptide synthesis. The optimized method, using 9-fluorenylmethoxycarbonyl protecting and singlestep coupling, yielded the crude product in 90% purity allowing for single-step reversed-phase HPLC purification to >98% purity and a high overall yield (50%). The hormone was characterized by several chromatographic methods, ion-spray mass spectroscopy and Edman degradation. The conformation of human neuropeptide Y was examined by CD, NMR and computer simulations. The CD measurements in trifluoroethanol/water (9: 1) show a large percentage of a-helix. Variation of concentration, from 0.5 pM increasing up to the 1 mM used for NMR measurements, indicates no evidence for aggregation. In the same solvent system, the NMR line widths were very broad and therefore the resonance assignment was achieved with the exclusive use of two-dimensional NOE spectra. The 248 clearly distinguishable NOES from the NMR study were used in distance geometry calculations and the resulting structures were refined with restrained molecular dynamics. The results indicate an a-helix extending from Argl9 to Gln34. For the N-terminal half of the molecule no regular structure was observed.Neuropeptide Y is a 36-amino-acid C-terminally amidated peptide isolated from porcine brain by Tatemoto et al. in 1982 [l]. It is widely distributed in the central and peripheral nervous system [2, 31. Within the central nervous system neuropeptide Y is involved in appetite [4], stress [5] and blood pressure [6]. In the peripheral nervous system neuropeptide Y has been shown to be a potent and long-lasting vasoconstrictor [7], acting as a cotransmitter and regulator at noradrenergic neurons [8]. Recent studies have indicated that neuropeptide Y has an analgesic effect [9].The primary structure of porcine neuropeptide Y has been determined by sequencing [lo] and synthesis [ll]. Human neuropeptide Y has a Met residue at position 17 instead of Leu. Earlier solid-phase syntheses of porcine neuropeptide Y were performed using Na-tert-butyloxycarbonyl (Boc) Abbreviutions. ADPV, 5-(4'-aminomethyl-3',5'-dimethoxyphenoxy)valeric acid; BOP, benzotriazol-1-yl-oxy-tris(dimethy1amino)-phosphonium hexafluorophosphate; Fmoc, 9-fluorenylmethoxycarbonyl; HOBt, 1-hydroxybenzotriazole; Mtr, 2,3,6-trimethyl-4-methoxysulfonylbenzene; NOESY, two-dimensional nuclear Overhauser effect spectroscopy; TBTU, 2-(I H-benzotriazol-1 -yl)-1 .I ,3,3-tetramethyluronium tetrafluoroborate; tBu, tert-butyl. benzotriazol-l-yl-oxy-tris(dimethylamino)-phosphonium hexafluorophosphate (BOP) activation and iV-Boc protection [18].A combined method was also described for the (1 -35)-peptide of human neuropeptide Y using solid-phase synthesis by diisopropylcarbodiimide and 1 -hydroxybenzotriazole (HOBt) activation and W-9-fluorenylmethoxycarbonyl (Fmoc) protection followed by enzymatic attachment of the C-terminal tyrosine...

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“…After the incorporation of ethylenediamine into the resin, Boc-E-aminocaproic acid (Boc-Aca), a 6-carbon spacer arm, was coupled to the support by using dimethyl formamide (DMF) as solvent. A 3-fold excess of Boc-Aca (7.2 mmol, 1.67 g) was used for the coupling and activation of the carboxylic function was achieved with BOP reagent (7.2 mmol, 3.18 g) in presence of DIEA (5-fold excess, 12 mmol, 1.55 g, 2.1 ml) (19).…”

mentioning

confidence: 99%

“…This compound was synthesized by the procedure of Mitchell et al (20). Boc-Ala-OMPA (1.8 mmol, 0.61 g) was coupled to the polymer by using the concomitant neutralizationcoupling step strategy (19,21) with BOP reagent (1.8 mmol, 0.79 g) and DIEA (2.7 mmol, 0.35 g, 0.47 ml).…”

mentioning

confidence: 99%

“…Before being treated with 40% trifluoroacetic acid in CH2Cl2, the Boc-Ala-resin was washed successively with DMF (two washes), isopropanol (two washes), and CH2Cl2 (two washes). Then, Boc-Leu28 and Boc-Gly27 were incorporated in the peptide chain by following the concomitant neutralization-coupling step protocol (19,21). After the deprotection of Gly27, the resin was washed with CH2Cl2 (three washes) and incubated for 2 min with 5% (vol/vol) DIEA in CH2Cl2.…”

mentioning

confidence: 99%

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Purification of a galanin receptor from pig brain.

Chen

Fournier

Couvineau

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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A galanin receptor protein was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) from pig brain membranes and then purified by single-step affinity chromatography. The product exhibits saturable and specific binding for galanin with a binding activity of 17 nmol/mg of protein and a dissociation constant (Kd) of 10 nM. This represents a 300,000-fold purification over the detergent-solubilized fraction with a final recovery of 31% of the initial membrane galanin binding activity. Gel electrophoresis of the affinity-purified material showed a single polypeptide of 54 kDa by silver staining and after radioiodination. Cross-linking of a purified fraction afrmity-labeled with 125I-labeled galanin revealed a single band for the galanin-receptor complex at 57 kDa. The general binding characteristics of the purified preparation appeared to be identical to those of the crude soluble material as far as specificity toward galanin and the structural requirement for galanin are concerned. In contrast, unlike the CHAPS-soluble galanin receptor, binding of 125I-labeled galanin to the purified galanin receptor was not sensitive to guanine nucleotides, suggesting that dissociation ofthe inhibitory guanine nucleotide binding protein from the galanin receptor occurred during purification. The purification to homogeneity of a galanin receptor paves the way toward its sequencing and cloning.Galanin is an ubiquitous neuropeptide identified in porcine intestine on the basis of its amidated C terminus (1). In agreement with its widespread localization in the central and peripheral nervous system (2), galanin elicits a wide range of biological responses (3). Since the discovery of galanin as a neuromodulator in the central nervous system (3, 4) and a sympathetic neuromodulator in the endocrine pancreas (5), its mechanism of action in these organs has been the focus of intensive study. Thus, radiolabeled galanin has been used to identify specific galanin binding sites mostly in the endocrine pancreas (6-10) and brain (11,12). In all these studies, the galanin receptor was shown to be coupled to a guanine nucleotide binding regulatory (G) protein to initiate the cascade of cellular responses through inhibition of adenylate cyclase (7, 13), activation of ATP-sensitive K+ channels (14, 15), or inhibition of calcium current (16). The molecular characterization of galanin receptors from brain and pancreas by covalent labeling with 125I-labeled galanin demonstrated that the galanin receptor is a monomeric glycoprotein of 53-54 kDa (6,8,10,11). Meanwhile, the functional association of the galanin receptor with an inhibitory G (Gi) protein was demonstrated (7,9,10,12,15 were from Bio-Rad; marker proteins were from BRL; disuccinimidyl tartarate (DST) was from Pierce; tert-butoxycarbonyl(Boc) amino acids and benzotriazolyl-N-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) were from Richelieu Biotechnologies (St-Hyacinthe, Quebec, Canada); fluoren-9-ylmethoxycarbonyl (Fmoc)-protected amino acids...

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Structural study of the N-terminal segment of neuropeptide tyrosine

Cited by 53 publications

References 5 publications

Characterization of melanotropin-release-inhibiting factor (melanostatin) from frog brain: homology with human neuropeptide Y.

Characterization of melanotropin-release-inhibiting factor (melanostatin) from frog brain: homology with human neuropeptide Y.

Neuropeptide Y

Purification of a galanin receptor from pig brain.

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