REVERSED-PHASE HPLC ANALYSIS OF PROENKEPHALIN-RELATED AND PRODYNORPHIN-RELATED END-PRODUCTS IN THE BRAIN OF A URODELE AMPHIBIAN, AMBYSTOMA-TIGRINUM

Acid extracts of the brain of a urodele amphibian, Ambystoma tigrinum, were screened with radioimmunoassays specific for enkephalin-related products and dynorphin-related products. Following Sephadex G-50 colum n chromatography a peak of enkephalin-sized immunoreactive material wa s detected near the total volume of the column. The enkephalin-sized i mmunoreactivity was further analyzed by reversed phase HPLC. This anal ysis detected peaks of authentic Met-enkephalin and Leu-enkephalin. Ho wever, the molar ratio of Met-enkephalin to Leu-enkephalin in the brai n of this amphibian was approximately 80:1. These observations would s uggest that the Leu-enkephalin detected in the brain of Ambystoma may be derived from a source other than the Pro-enkephalin precursor. Neit her Met-enkephalin-RGL or Met-enkephalin-RF were detected by radioimmu noassay in brain extracts from this urodele. However, following digest ion with trypsin and carboxypeptidase B, a novel peak of C-terminally extended Met-enkephalin was detected. Two peaks of Prodynorphin-relate d products were also detected following gel filtration chromatography. These immunoreactive forms were detected using antisera specific for alpha-neo-endorphin and dynorphin B(1-13). No immunoreactive forms wit h antigenic determinants similar to mammalian dynorphin A(1-17) or dyn orphin A(1-8) were detected in this species. Reversed phase HPLC analy sis indicated that the major form of urodele alpha-neo-endorphin elute d with the same. retention time as synthetic mammalian alpha-neo-endor phin. Urodele dynorphin B(1-13)-related immunoreactivity eluted as a s ingle peak, however this form did not elute with the same retention ti me as synthetic mammalian dynorphin B(1-13). The forms of alpha-neo-en dorphin and dynorphin B detected in urodele and anuran amphibians appe ar to be very similar, however it appears that amphibian dynorphin A h as diverged significantly from mammalian dynorphin A. Analysis of enke phalin-related products in the brain of Ambystoma indicates that Met-e nkephalin is the major opioid produced by the urodele Proenkephalin ge ne, and supports the contention that urodele Proenkephalin, like anura n Proenkephalin, lack a Leu-enkephalin sequence.