Isolation, primary structure, and synthesis of α-endorphin and γ-endorphin, two peptides of hypothalamic-hypophysial origin with morphinomimetic activity

Ling, Nicholas; Burgus, Roger; Guillemin, Roger

doi:10.1073/pnas.73.11.3942

Cited by 184 publications

(43 citation statements)

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“…Rossier, R. Guillemin, and F. Bloom, personal communication), it is unlikely that the immunofluorescence observed in the present study is attributable to these endorphins. These larger opioid peptides occur in very high concentrations in the pituitary (24)(25)(26).…”

mentioning

confidence: 99%

Opioid peptide enkephalin: immunohistochemical mapping in rat central nervous system.

Simantov¹,

Kuhar²,

Uhl³

et al. 1977

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

450

105

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Using specific antisera to methionine-enkephalin and leucine-enkephalin, we have visualized apparent enkephalin-containing neuronal fibers and terminals throughout the central nervous system of the rat. Immunoreactive enkephalin displays sharply defined localizations. Regions of highest immunofluorescent density include the laminae I and II of the spinal cord, the substantia gelatinosa of the caudal nucleus of nerve V, the vagal nuclei of the medulla, the periventricular and periaqueductal areas of the upper medulla and midbrain, dorsomedial thalamic regions, specific hypothalamic nuclei, the basal ganglia, particularly the globus pallidus and the central nucleus of the amygdala, and the lateral septum. In certain regions enkephalin immunofluorescence corresponds closely with the distribution of autoradiographic opiate receptor grains.The opiate-like pentapeptides methionine-enkephalin (Metenk) and leucine-enkephalin (Leu-enk) (1, 2) appear to be endogenous ligands for the opiate receptor. The regional localization of enkephalin in mammalian brain, determined biochemically, resembles that of opiate receptor binding (3-6). In subcellular fractionation studies, enkephalin is localized to synaptosomal fractions that contain nerve terminals (7). Autoradiographic studies of the opiate receptor reveal sharply defined localizations to structures mediating functions affected by opiates, such as pain perception (8)(9)(10). If the enkephalins are neurotransmitters or neuromodulators associated with opiate receptors, one might expect enkephalin to be localized microscopically to neuronal systems impinging on opiate receptors. Preliminary immunohistochemical studies show immunoreactive enkephalin fluorescence in nerve fibers and terminals with highest densities in areas enriched in opiate receptors (11). We now report a detailed mapping of the rat central nervous system for immunoreactive enkephalin.MATERIALS AND METHODS Antisera Preparation. Met-enk or Leu-enk (20 mg) was coupled to keyhole limpet hemocyanin (10 mg) by incubation for 30 min in distilled water at room temperature with 150 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The material was dialyzed extensively against distilled water, lyophilized, suspended in 3 ml of distilled water, and stored. Guinea pigs were immunized with 1 mg of the hemocyanincoupled enkephalin diluted 1:10 in saline and mixed 1:1 with Freund's complete adjuvant. Rabbits were injected with 1.6 mg of this conjugate. The immunization was repeated three to four times at 3 to 4 week intervals using incomplete Freund's adjuvant. The guinea pigs and rabbits were bled 7-10 days after the third and subsequent immunizations and the sera were tested for enkephalin binding in radioimmunoassays (ref. 12; Abbreviations: Met-enk, methionine-enkephalin; Leu-enk, leucineenkephalin.* To whom reprint requests should be addressed.Simantov, Childers, and Snyder, unpublished data). Enkephalin binding by these antisera was not displaced by high concentrations of substance P, glucagon, insulin...

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mentioning

confidence: 99%

Opioid peptide enkephalin: immunohistochemical mapping in rat central nervous system.

Simantov¹,

Kuhar²,

Uhl³

et al. 1977

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

450

105

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“…ments of ACTH (ACTHI-o, ACTH410, and a-MSH) have been found to have central nervous system effects (7) whereas fragments of ,B-LPH [0-endorphin (3-LPH61-91), a-endorphin (f-LPH61 70), and enkephalin (f3-LPH61-65)] appear to have opiate-like activity (8)(9)(10). The recent implications of the physiological significance of these peptides in the nervous system stimulated great interest in their biosynthesis, particularly in determining if they are cleaved from ACTH and ,B-LPH.…”

mentioning

confidence: 99%

Immunological evidence for two common precursors to corticotropins, endorphins, and melanotropin in the neurointermediate lobe of the toad pituitary.

Loh¹

1979

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

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The biosynthesis of corticotropin (ACTH1-39), and a-melanotropin in the toad intermediate lobe was studied by using immunoprecipitation procedures with antisera specific for these peptides. Intermediate lobes were pulse-incubated with [3H phenylalanine and then chase-incubated for varying periods; the radioactive proteins were immunoprecipitated. Immunoprecipitates were separated by acidic urea or sodium dodecyl sulfate polyacrylamide gel electrophoresis. Evidence from the pulse-chase and sequential immunoprecipitation studies using antisera to ACTH and 3-endorphin suggests that the toad intermediate lobe synthesizes two common precursors (apparent M, 32,000 and 29,500) containing both the ACTH and 3-endorphin sequences. from mouse appear to synthesize not only ACTH but also ,B-LPH from cleavage of a common precursor of approximately 31,000 daltons. In addition, a polyadenylylated RNA fraction from such tumor cells has been translated in the reticulocyte and wheat germ cell-free systems and, among the translation products, is a 28,500-dalton protein that is precipitated by anti-ACTH antisera and includes tryptic digestion sequences that are indistinguishable from those of ACTH and ,15 After the pulse, the neurointermediate lobes were homogenized. A group of 0.5-hr pulsed lobes were subjected to a chase in amphibian Ringer's solution containing unlabeled phenylalanine (1 mM) for 1 or 3 hr before homogenization. The medium after a 3-hr chase was also collected for analysis of released peptides. Lobes were homogenized in 5 M acetic acid containing 5 mg of bovine serum albumin per ml and 1% phenylmethylsulfonyl fluoride (30 mg/ml)/iodoacetamide (30 mg/ml) in absolute ethanol, freshly made within hours of its use. Samples were frozen and thawed three times on dry ice and extracted at 4°C for 12-16 hr. Extracts were then lyophilized.Abbreviations: LPH, lipotropin; ACTH, corticotropin; MSH, melanotropin; NaDodSO4, sodium dodecyl sulfate. 796The 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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“…al., Ling et al, 1976). These peptides have been designated ct-endorphin (fl-endorphin-(1-16)) and y-endorphin (/~-endorphin-(1-17)) (Fig.…”

Section: Fl-endorphin Fragments: ~-And 7-endorphinsmentioning

confidence: 99%

Action of proteolytic enzymes on lipotropins and endorphins: Biosynthesis, biotransformation and fate

Burbach¹

1984

Pharmacology & Therapeutics

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Isolation, primary structure, and synthesis of α-endorphin and γ-endorphin, two peptides of hypothalamic-hypophysial origin with morphinomimetic activity

Cited by 184 publications

References 20 publications

Opioid peptide enkephalin: immunohistochemical mapping in rat central nervous system.

Opioid peptide enkephalin: immunohistochemical mapping in rat central nervous system.

Immunological evidence for two common precursors to corticotropins, endorphins, and melanotropin in the neurointermediate lobe of the toad pituitary.

Action of proteolytic enzymes on lipotropins and endorphins: Biosynthesis, biotransformation and fate

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