The synthesis and release of [3H-tyrosine1]methionine5-enkephalin from guinea pig brain slices.
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.
D000818:Animals; D001285:Atropine / Q000494:pharmacology; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D004744:Enkephalin, Methionine / Q000096:biosynthesis* / Q000378:metabolism; D006168:Guinea Pigs; D066298:In Vitro Techniques; D007700:Kinetics; D011188:Potassium / Q000494:pharmacology; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism*; D014316:Tritium; D014443:Tyrosine / Q000378:metabolism; D014701:Veratridine / Q000494:pharmacology
D014316:Tritium; D014443:Tyrosine; D004744:Enkephalin, Methionine; D014701:Veratridine; D001285:Atropine; D002217:Carbachol; D011188:Potassium