RADIOIMMUNOASSAY OF METHIONINE-AND LEUCINE-ENKEPHALINS IN REGIONS OF RAT BRAIN AND COMPARISON WITH ENDORPHINS ESTIMATED BY A RADIORECEPTOR ASSAY

Abstract— Radioimmunoassays (RIAs) selective for methionine-enkephalin (Met-ENK) and leucineenkephalin (Leu-ENK) have been developed using competition towards binding of 10 pM ¹²⁵I-enkephalins to antibodies raised in rabbits against ENKs coupled to ovalbumin with carbodiimide. The high sensitivity of the RIAs (IC₅₀ 0.57 n m 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 n m ³H]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 -HCl 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 2–13% of the total endorphins (RRA) suggesting the presence of large amounts of endorphins other than the ENKs.