Separation and partial characterization by high-performance liquid chromatography and radioimmunoassay of different forms of melanocyte-stimulating hormone from fish (Cyprinidae) neurointermediate lobes

Abstract

From neurointermediate lobe (NIL) extracts of two species of Cyprinidae, Carassius auratus and Cyprinus carpio, several peptides were separated by high-performance liquid chromatography (HPLC) on a C18 muBondapak column eluted with a methanol/acetic acid/triethylamine mixture. Monitoring all fractions by radioimmunoassay (RIA) with an antibody against melanocyte-stimulating hormone (MSH) C terminal gave positive reactions for fractions 7, 11-12, 15-16, 23-24, and 25-27. For further characterization, the elution positions of these peaks were compared to those of known synthetic reference substances. Peak 7 elutes in the same position as oxidized alpha MSH, whereas peak 15-16 matches the elution position of des-acetyl alpha MSH and 23-24 that of alpha MSH. The product from peak 26-27 has several characteristics of the diacetylated form of alpha MSH: its immunoreactivity in RIA, its sensitivity to weak bases and to HCl and its mass spectrum which is identical with that of mammalian diacetyl alpha MSH. In both species, the diacetylated form is predominant in the intracellular pool. This study establishes the coexistence of three different forms of alpha MSH, a des-acetylated, monoacetylated, and diacetylated in the cyprinid NIL extracts.