Isolation and characterization of two coho salmon gonadotropins, GTH I and GTH II

Abstract

Two gonadotropins, GTH I and GTH II, were isolated from pituitaries of spawning coho salmon (Oncorhynchus kisutch) using sequential extractions with ammonium acetate (pH 9.0) and 40% ethanol, precipitation with 80% ethanol, gel filtration chromatography (Sephadex G-100), anion-exchange chromatography (Mono-Q Sepharose), and gel filtration chromatography (Sephadex G-75). Coho salmon GTH I and GTH II stimulated steroidogenesis in vitro in a similar dose-dependent manner when incubated with either ovaries or testes of prepubertal coho salmon. An in vivo bioassay using coho salmon parr demonstrated that coho salmon GTH I and GTH II did not contain thyrotropic activity. Molecular weights were estimated by gel filtration chromatography to be 43 000 and 39 000 for GTH I and GTH II, respectively. Analysis of coho salmon GTH I and GTH II on reversed-phase high-performance liquid chromatography (rpHPLC) revealed that they consist of α and β subunits with N-terminal amino acid residues of Tyr, Gly (α,β of GTH I) and Tyr, Ser (α,β of GTH II). Coho salmon GTH I-β and GTH II-β differed from each other in amino acid composition, N-terminal amino acids (Gly vs. Ser), and molecular weights in SDS-PAGE (19 000 vs. 20 000) and had a high degree of chemical similarity to chum salmon GTH I-β and GTH II-β, respectively. Specific rabbit antisera to the β subunits of coho salmon GTH I and GTH II were generated. The observation of two GTHs with distinctly different chemical characteristics in coho salmon is similar to what has previously been found in chum salmon.