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  • Title: The synthesis and release of [3H-tyrosine1]methionine5-enkephalin from guinea pig brain slices.
    Author: Jones CA, Marchbanks RM.
    Journal: J Neurochem; 1983 Feb; 40(2):357-63. PubMed ID: 6822829.
    Abstract:
    Stores of methionine-enkephalin were labelled on the N-terminal by incubation of whole brain slices with [3H]tyrosine (10 microCi/ml). The 3H radioactivity corresponding to the position of authentic Met-enkephalin after extraction on Amberlite XAD2 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 degrees C and was inhibited in the presence of 10 microM cycloheximide. Isolated nerve terminals failed to incorporate any [3H]tyrosine. The labelled compound had opiate-like 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 [14C]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 mM) elicited a Ca2+-dependent release of the synthesized [3H]Met-enkephalin from whole brain slices and also from isolated nerve terminals. The release of Met-enkephalin radioimmunoactivity paralleled that of [3H]met-enkephalin. Preliminary investigations have suggested that carbamyl choline inhibited this release, and its effect was partially reversed by atropine.
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