Forms of immunoreactive β-endorphin in the intermediate pituitary of the holostean fish, Amia calva

Abstract

Acid extracts of the intermediate pituitary of the holostean fish, Amia calva, were fractionated by gel filtration chromatography and analyzed with radioimmunoassays specific for N-acetylated β-endorphin and C-terminally amidated α-MSH. In these extracts β-endorphin-related immunoreactive material and α-MSH-related immunoreactive material were present in roughly equimolar amounts. The immunoreactive β-endorphin-sized material was tested for opiate receptor binding activity using a β-endorphin radioreceptor assay. The results of these studies were negative. The immunoreactive β-endorphin-sized material was further analyzed by cation exchange chromatography at pH 2.5. Two major and three minor peaks of immunoreactive material were isolated. Peak 5 exhibited a net charge of +7 at pH 2.5 and represented 53% of the total immunoreactivity recovered. Peak 2 with a net charge of +3 at this pH represented 38% of the total immunoreactivity recovered. The minor forms, Peaks 1, 3 and 4, exhibited net charges of +2, +4 and +6, respectively. The apparent molecular weights of Peaks 2 and 5 were determined on a Sephadex G-50 column. Peak 2 had an apparent molecular weight of 2.7 Kd and Peak 5 had an apparent molecular weight of 3.5 Kd. Reverse phase HPLC analysis of Peak 5 indicates that this form of Amia β-endorphin had chromatographic properties similar to salmon β-endorphin II. These results would suggest that N-terminal acetylation and C-terminal proteolytic cleavage are important post-translational modifications of the forms of Amia β-endorphin.