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Radioimmunoassays (RIAs) selective for methionine-enkephalin (Met-ENK) and leucineenkephalin (Leu-ENK) have been developed using competition towards binding of 10 pM 1*51-enkephalins to antibodies raised in rabbits against ENKs coupled to ovalbumin with carbodiimide. The high sensitivity of the RlAs (ICso 0.57 nM and 0.55 n M for Met-and Leu-ENK, respectively) allowed estimation of the enkephalin content in extracts of all rat brain regions. Regional levels are compared with those determined on the same extracts by a radioreceptor assay (RRA) using competition towards binding of 5 nM [)H]Leu-ENK to rat striatal membranes. Optimal conditions for killing the animals and extracting the endorphins have been carefully investigated: killing by rapid microwave irradiation was not found necessary as long as brain regions were homogenized into 0.1 N-HCI before deproteinization.Marked differences both in total endorphins (RRA) and ENKs (RIA) between regions are observed with similar ranking of the various regions: highest levels are found in striatum and hypothalamus and lowest in cerebellum and hippocampus. In each region the total ENK levels (RIA) represent only 2-1396 of the total endorphins (RRA) suggesting the presence of large amounts of endorphins other than the ENKs HUGHES (1975) has identified in brain extracts an endogenous ligand for the opiate receptor which mimics the action of morphine in the mouse vas deferens or the guinea-pig ileum. He has suggested that the substance could be a small peptide. HUGHES et al. (1975) then isolated opioid peptides from pig brain and these were identified as a mixture of two pentapeptides termed methionine-enkephalin (Met-ENK; H-Tyr-Gly-Gly-Phe-Met-OH) and leucineenkephalin (Leu-ENK; H-Tyr-Gly-Gly-Phe-Leu-OH). The aminoacid sequence of the two enkephalins has been confirmed by SIMANTOV & SNYDER (1976a). Met-ENK corresponds to a fraction (as residues 61-65) of a pituitary peptide containing 91 aminoacids, b-lipotropin.Independently. the presence of other endorphins. i.e. endogenous peptides that mimic the actions of morphine, has been characterized in pituitary extracts ' Maitre de Recherches a 1'I.N.S.E.R.M.

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