Immunological evidence for multiple forms of alpha-melanotropin (alpha-MSH) in the pars intermedia of the Australian lungfish, Neoceratodus forsteri.
Acid extracts of individual pars intermedia from the Australian lungfish, Neoceratodus forsteri, were fractionated by gel filtration chromatography and analyzed for alpha-melanotropin (alpha-MSH) immunoreactivity. In these studies a C-terminal-specific alpha-MSH radioimmunoassay (RIA) was used. Following gel filtration chromatography on a Sephadex G-75 column, a major peak of immunoreactive alpha-MSH-sized material was detected. On the average there was 338 +/- 72 pmol (SD) of immunoreactive alpha-MSH per lungfish pars intermedia (n = 3). Following gel filtration the immunoreactive alpha-MSH was further analyzed by reverse-phase high-performance liquid chromatography (HPLC). Three peaks of immunoreactivity were detected. These peaks were designated Peaks 1, 2, and 3. The retention times of these peaks corresponded to, respectively, mammalian ACTH(1-13)amide, N-acetyl-ACTH(1-13)-amide, and N,O-diacetyl-ACTH(1-13)amide. Peaks 2 and 3 represented approximately 95% of the immunoreactive alpha-MSH recovered. Analysis of immunoreactive Peaks 2 and 3 by cation-ion-exchange indicated that both peaks had a net charge of +3 at pH 2.5. Since O-acetyl groups are sensitive to high pH, Peak 3 was incubated for 1 hr at 37 degrees in 0.01 N NaOH, pH 12. Under these conditions, Peak 3 eluted with the same retention time as untreated Peak 2. Collectively, these results indicate that Peaks 2 and 3 correspond to mono- and diacetylated lungfish alpha-MSH, respectively.
Dores
R M
RM
University of Denver, Department of Biological Sciences, Colorado 80208.
Joss
J M
JM
D000818:Animals; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange; D005399:Fishes / Q000378:metabolism*; D010902:Pituitary Gland / Q000032:analysis*; D011863:Radioimmunoassay; D000521:alpha-MSH / Q000032:analysis*
D000521:alpha-MSH