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The Synthesis and Release of [ 3 H‐Tyrosine 1 ]Methionine 5 ‐Enkephalin from Guinea Pig Brain Slices
Author(s) -
Jones C. A.,
Marchbanks R. M.
Publication year - 1983
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1983.tb11290.x
Subject(s) - tyrosine , chemistry , enkephalin , incubation , leucine , choline , methionine , biochemistry , amino acid , chromatography , stereochemistry , receptor , opioid
Stores of methionine‐enkephalin were labelled on the N ‐terminal by incubation of whole brain slices with [ 3 H]tyrosine (10 °Ci/ml). The 3 H radioactivity corresponding to the position of authentic Met‐enkephalin after extraction on Amberlite XAD 2 and separation by thin‐layer chromatography was taken as an index of synthesis. Maximal incorporation of the labelled tyrosine into Met‐enkephalin was attained after 4 h of incubation at 37°C and was inhibited in the presence of 10 μ M cycloheximide. Isolated nerve terminals failed to incorporate any [ 3 H]tyrosine. The labelled compound had opiatelike activity and consisted of the same five amino acids as an authentic standard. Incubations with leucine aminopeptidase indicated that the labelled tyrosine was on the N ‐terminus and removal of this tyrosine resulted in loss of opiate‐like activity. The incorporation of [ 14 C]glycine, selected as an alternative precursor, was consistent with de novo synthesis and not N ‐terminal exchange. A radioimmunoassay was also used to quantify the amount of labelled Met‐enkephalin. KCl (50 m M ) elicited a Ca 2+ ‐dependent release of the synthesised [ 3 H]Met‐enkephalin from whole brain slices and also from isolated nerve terminals. The release of Met‐enkephalin radioimmunoactivity paralleled that of [ 3 H]met‐enkephalin. Preliminary investigations have suggested that carbamyl choline inhibited this release and its effect was partially reversed by atropine.