The isolation of multiple forms of beta-endorphin from the intermediate pituitary of the Australian lungfish, Neoceratodus forsteri

The pituitary of the Australian lungfish, Neoceratodus forsteri, was screened immunohistochemically with heterologous antisera specific for either the C-terminal of mammalian beta-endorphin or the acetylated N-terminal of beta-endorphin. Immunopositive cells were only detected with the N-terminal specific antiserum; these cells were restricted to the intermediate pituitary. Acid extracts of the intermediate pituitary were fractionated by Sephadex gel filtration chromatography, CM cation exchange chromatography and reverse phase HPLC. Fractions were analyzed by radioimmunoassay (RIA) with a N-acetyl specific beta-endorphin RIA and by radioreceptor assay for the presence of opiate active forms of beta-endorphin. Both immunoreactive and opiate active forms of beta-endorphin were detected. Of the total beta-endorphin-related material isolated from the intermediate pituitary, approximately 97% was detected with the N-terminal specific RIA and approximately 3% was detected by the radioreceptor assay. The N-acetylated immunoreactive beta-endorphin could be separated into two forms. The major form had an apparent molecular weight of 3.2 Kda. This material had a net charge at pH 2.5 of +5. The minor form of immunoreactive beta-endorphin had an apparent molecular weight of 1.4 Kda and a net charge at pH 2.5 of +1. Neither immunoreactive form exhibited receptor binding activity in the radioreceptor assay. A single peak of opiate active beta-endorphin was detected. This material had an apparent molecular weight of 3.5 Kda and a net charge at pH 2.5 of +7.