Purification, Characterization, and Bioassay of Prolactin and Growth Hormone from Temperate Basses, Genus Morone

Abstract Prolactin (PRL) and two variants of growth hormone (GH), purified from pituitaries of striped bass ( Morone saxatilis ) and its hybrid with white bass ( M. saxatilis × M . chrysops ) by gel filtration chromatography under alkaline conditions followed by reversed-phase high pressure liquid chromatography, appear similar between species. Both the minor (GH I) and the major (GH II) forms of purified GH appeared as single bands ( M r ∼ 23,000) after sodium dodecyl sulfate–polyacrylamide gel electrophoresis, as did the purified PRL ( M r ∼ 24,000). The molecular weights of GH II and PRL determined by MALDI TOF mass spectroscopy were 21.2 and 21.3 kDa, respectively. In Western blotting experiments, an antiserum against tilapia ( Oreochromis mossambicus ) 24K PRL specifically recognized Morone PRL, while an antiserum against tilapia GH specifically recognized Morone GH I and II. Chemical identities of the putative PRL and GH I were further confirmed by N-terminal peptide sequencing, while internal sequence analysis was performed on GH II because it was blocked at its N-terminus. Over a stretch of 29 amino acids, Morone PRL was found to be 76% identical to tilapia 24K PRL, 72% identical to tilapia 20K PRL, 72% identical to chum salmon ( Oncorhynchus keta ) PRL I, and 69% identical to eel ( Anguilla japonica ) PRL I. Alignment of the hybrid striped bass GH sequences with those of several other advanced marine teleosts indicated 75–85% sequence identity for GH I (40 amino acids) and 95–98% identity for GH II (45 amino acids). Biological activity of striped bass GH II was confirmed using a heterologous in vitro assay of insulin-like growth factor I mRNA production by coho salmon ( On . kisutch ) hepatocytes. An in vivo bioassay, involving hypophysectomy of hybrid striped bass and treatment of the fish maintained in fresh water with homologous PRL, confirmed that the purified striped bass PRL was also bioactive.