Isolation and structure of pro-methionine-enkephalin: Potential enkephalin precursor from porcine hypothalamus

Huang, Wei; Chang, Robert C.; Kastin, Abba J.; Coy, David H.

doi:10.1073/pnas.76.12.6177

Cited by 49 publications

(12 citation statements)

References 23 publications

(12 reference statements)

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“…The differential effects of ions on agonist and antagonist binding also were shown to be similar to those observed in mammalian brain homogenates. These earlier studies tentatively indicated that the opiate sites are analogous to the enkephalin-type (8) receptor.…”

Section: Discussionmentioning

confidence: 90%

“…The present study not only supports these immunocytochemical observations but also di- The occurrence of both [Met]-and [LeuJenkephalin in M. edulis pedal ganglia was anticipated due to a number of reports that indirectly suggested the presence of some types of opioid compounds. Etorphine, FK 33-824, and levallorphan bind noncooperatively to a class of high-affinity sites (Kd = 1-3 nM) and cooperatively to a class of lower-affinity sites (Kd = 6-11 nM) in the pedal ganglion (8). Hill analysis of the cooperative sites revealed Hill coefficients of 2.6-3.7, indicating markedly positive homotropic cooperativity.…”

Section: Discussionmentioning

confidence: 99%

“…An aliquot was then subjected to HPLC on a Brownlee RP-300 reverse-phase column (4.6 x 250 mm). The column was eluted at a flow rate of 2 ml/min with a solvent system consisting of the HPLC buffer and a linear gradient of 5-25% (vol/vol) 2-propanol in 15 min (8).…”

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confidence: 99%

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Isolation and identification of enkephalins in pedal ganglia of Mytilus edulis (Mollusca).

Leung¹,

Stefano²

1984

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

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An acid extract of pedal ganglia of the mollusc Mytilus edulis was fractionated by high-pressure liquid chromatography with a reverse-phase column. Peak fractions with retention times of those of [Met] MATERIALS AND METHODS Acid Extraction. The acid extraction of low molecular weight peptides from mussel pedal ganglia was carried out as described (7). The ganglia were homogenized with a Brinkmann Polytron homogenizer with four 3-sec bursts in an extraction solution (25 pedal ganglia per ml) containing 1 M acetic acid, 20 mM HCl, 1 ,ug each of phenylmethyl-sulfonyl fluoride and pepstatin A per ml, and 1% 2-mercaptoethanol. The homogenate was clarified by centrifugation at 27,000 x g for 1 hr. The supernatant was then deproteinized by the addition of 50% (wt/vol) trichloroacetic acid to reach a final concentration of 10% (wt/vol) and centrifuged at 27,000 x g for 30 min. The lipids and trichloroacetic acid in the supernatant were removed by extracting three times with equal volumes of diethyl ether. The aqueous portion was lyophilized and stored. All of these steps were carrie4 out at 40C.High-Pressure Liquid Chromatography (HPLC). All steps subsequent to extraction were carried out at room temperature. The lyophilized extract was dissolved in a buffer (one pedal ganglion per 41 consisting of 10 mM ammonium acetate, pH 4.0. The HPLC system used was a Beckman model 334 liquid chromatograph equipped with a Beckman/Altex 210 sample injector and a Beckman/Altex C-RIA integrator. Before injection, the sample was clarified by centrifugation at 1,000 x g for 10 min. An aliquot was then subjected to HPLC on a Brownlee RP-300 reverse-phase column (4.6 x 250 mm). The column was eluted at a flow rate of 2 ml/min with a solvent system consisting of the HPLC buffer and a linear gradient of 5-25% (vol/vol) 2-propanol in 15 min (8).For the isolation of crude fractions the highly polar components of the extract were removed by an initial isocratic elution with 5% (vol/vol) 2-propanol for 19 min before the gra- Sound at Northport, NY. For binding studies, pedal ganglia from fresh M. edulis were processed as described (9). Immediately prior to the binding experiment, the supernatant was centrifuged at 30,000 x g for 15 min, and the resulting pellet was washed with 50 vol of sucrose/Tris-HCl buffer. The Tris'HCl buffer used was 150 mM KCI in 10 mM Tris HCl buffer, pH 7.4, supplemented with 0.1% bovine serum albumin. Binding assays were carried out by a modification of the method of Pert and Snyder (10) 955The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Section: Discussionmentioning

confidence: 90%

Section: Discussionmentioning

confidence: 99%

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Isolation and identification of enkephalins in pedal ganglia of Mytilus edulis (Mollusca).

Leung¹,

Stefano²

1984

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

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“…Similarly, the a-MSH did not inhibit the contractions of the mouse vas deferens at 1 MM (12). When assayed for lipolytic activity, this decapeptide failed to stimulate an increase in glycerol production in doses 4 to 4000 times greater than the effective lipolytic dose of ACTH in our system (17).…”

Section: Resultsmentioning

confidence: 99%

“…The extract corresponding to 470,000 hypothalami was the same as that used for the isolation of porcine somatostatin (3) and pro- [Met]enkephalin (4). This extract was purified first by gel filtration on Sephadex G-25 (3).…”

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confidence: 99%

Isolation, structure, biological characterization, and synthesis of beta-[Tyr9]melanotropin-(9-18) decapeptide from pig hypothalami.

Schally¹,

Chang²,

Huang³

et al. 1980

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

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A decapeptide with the amino acid sequence H-Tyr-Phe-Arg-Trp-Gly-Ser-Pro-ProLys-Asp-OH was isolated from acid extracts of porcine hypothalami, structurally and biologically characterized, and synthesized. Except for the NH2-terminal tyrosine, this decapeptide corresponds to the amino acid sequences 9-18 of porcine ,Bmelanotropin (P-MSH) and 49-58 of porcine fl-ipotropin (#-LPH) it also has a tetrapeptide sequence of amino acids (Phe-Arg-Trp-Gly) common to the 7-10 sequences in corticotropin (ACIT) and a-MSH. P-MSH, P-LPH, a-MSH, and ACTIH from various species all have a histidine residue in the position immediately preceding the common sequence, and the occurrence of a natural peptide with the tyrosine residue in the corresponding site has not been previously reported. This suggests that the fTyMSH49-18) decapeptide might be a fragment of a still larger precursor (prohormone) possibly related to ,-LPH.During our ongoing project on characterization and systematic classification of peptides from the diencephalon (1-5), we isolated a decapeptide from porcine hypothalami which has a COOH-terminal nonapeptide portion identical with amino acids 10-18 of porcine f3-melanotropin (f3-MSH) and 50-58 of f3-lipotropin (f3-LPH) but which, in contrast to these two polypeptides, has tyrosine instead of histidine directly preceding the common sequence. This paper describes the isolation, elucidation of structure, synthesis, and biological characterization of this decapeptide, which could possibly be a cleavage fragment of a larger precursor.MATERIALS AND METHODS Purification. The extract corresponding to 470,000 hypothalami was the same as that used for the isolation of porcine somatostatin (3) and pro-[Met]enkephalin (4). This extract was purified first by gel filtration on Sephadex G-25 (3). After extraction with phenol, the material was purified by chromatography on CM-cellulose, counter current distribution in solvent system I (0.1% acetic acid/1-butanol/pyridine, 11:5:3, vol/vol) in the A-4 apparatus, chromatography and rechromatography on SE-Sephadex, analytical gel filtration on Sephadex G-15, and finally partition chromatography in solvent system II (1-butanol/1-propanol/acetic acid/water, 7:1:2:10, vol/vol). In all steps, the separation pattern was followed by the Folin-Lowry reaction (6) or by absorbance at 278 nm.Edman Dansyl Procedure. The Edman degradation was carried out according to the procedure described by Hartley (7). Dansyl amino acids were identified by thin-layer chromatography (TLC) on polyamide sheets (7). Purification of tryptic fragments and comparison of the natural and synthetic materials were carried out by high-pressure liquid chromatography (HPLC). All the extraction and purification procedures and methods used for homogeneity and composition determinations and for the manual Edman dansyl degradation have been described in detail (1-5).Aminopeptidase (9) or isolated rat pituitary quarters (2, 3, 10, 11). Each sample was assayed in quadruplicate. The concentration of pituitary hormones in...

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Localization of enkephalin‐like immunoreactivity to identified axonal and neuronal populations of the rat hippocampus

Gall

Brecha

Karten

et al. 1981

J of Comparative Neurology

345

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The distribution of enkephalin-like immunoreactivity in the hippocampal formation of the rat was analyzed. Two specific projection systems are described. The first emerges from the hilus of the dentate gyrus and appears to terminate with notably large boutons on the proximal apical and, to a lesser extent, basal dendrites of hippocampal regio inferior pyramidal cells. This projection corresponds in source, position, and character to the hippocampal mossy fiber system. The second axonal population enters the temporal hippocampal formation from the medial wall of the subicular complex and follows the hippocampal fissure to occupy stratum lacunosum-moleculare of the hippocampus proper and the distal third of the dentate gyrus molecular layer; this pattern corresponds to the distribution of afferent input from the lateral entorhinal cortex and/or perirhinal area. Lesions of the hilus or retrohippocampal area caused a selective depletion of immunoreactivity in the mossy fiber fields and molecular layers of the dentate gyrus, respectively. Enkephalin-like immunoreactivity was found within the somata of three types of hippocampal neurons: 1) granule cells of the dentate gyrus, 2) occasional pyramidal shaped cells of field CA1 stratum pyramidale, and 3) varied scattered interneurons. Of this last group, two types of interneurons were consistently seen. The first occupy the border between stratum radiatum and stratum lacunosum-moleculare and extend processes at right angles to the long axis of the pyramidal cell dentrites, whereas the second lie within stratum radiatum of field CA1 and extend processes in alignment with the long axis of the pyramidal cell dendrites. Cells containing enkephalin-like immunoreactivity were also observed in the subiculum and retrohippocampal region, most notably including layers II and III of the lateral entorhinal cortex-perirhinal area--the probable source of extrinsic immunoreactive input to the hippocampal formation. Intraventricular colchicine treatment intensified the immunoreactive staining of some hippocampal neurons but did not reveal any cell types not seen to be labeled in untreated rats.

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Isolation and structure of pro-methionine-enkephalin: Potential enkephalin precursor from porcine hypothalamus

Cited by 49 publications

References 23 publications

Isolation and identification of enkephalins in pedal ganglia of Mytilus edulis (Mollusca).

Isolation and identification of enkephalins in pedal ganglia of Mytilus edulis (Mollusca).

Isolation, structure, biological characterization, and synthesis of beta-[Tyr9]melanotropin-(9-18) decapeptide from pig hypothalami.

Localization of enkephalin‐like immunoreactivity to identified axonal and neuronal populations of the rat hippocampus

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