Immunohistochemical and biochemical evidence for the presence of the pentapeptide met-enkephalin and the heptapeptide met-enkephalin-Arg(6)-Phe(7) but not the octapeptide met-enkephalin-Arg(6)-Gly(7)-Leu(8) in amphibian chromaffin cells.

Using specific antibodies to met-enkephalin, met-enkephalin-Arg(6)-Phe(7) and met-enkephalin-Arg(6)-Gly(7)-Leu(8), we have studied the distribution of these opioid peptides in the frog adrenal gland by means of the indirect immunofluorescence technique. Bright staining of all chromaffin cells was observed by application of met-enkephalin and met-enkephalin-Arg(6)-Phe(7) antisera. No nerve endings could be detected. A few chromaffin cells were weakly stained by met-enkephalin-Arg(6)-Gly(7)-Leu(8) antiserum. Using a specific radioimmunoassay for met-enkephalin, the dilution curve of frog adrenal extracts was parallel to that of synthetic met-enkephalin. The concentration of met-enkephalin-like material measured in crude acetic extracts of frog adrenals (2.31 +/- 0.16 pmol/mg w. wt) was high when compared to those reported for most mammalian species. No leu-enkephalin and virtually no met-enkephalin-Arg(6)-Gly(7)-Leu(8) were detected by the corresponding radioimmunoassays. Reverse phase HPLC analysis revealed that oxidized met-enkephalin was the main form of met-enkephalin detected in acetic extracts of frog adrenals. HPLC separation showed the presence of a peptide co-eluting with synthetic met-enkephalin-Arg(6)-Phe(7). Higher molecular weight forms were also separated by HPLC. These results show the presence of both met-enkephalin and the heptapeptide met-enkephalin-Arg(6)-Phe(7) and the lack of met-enkephalin-Arg(6)-Gly(7)-Leu(8) in frog chromaffin cells.