Isolation and characterization of two distinct gonadotropins, GTHI and GTHII, from bonito (Katsuwonus plelamis) pituitary glands

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Abstract
Two distinct glycoproteins homologous to chum salmon GTHI and GTHII were isolated from the pituitary glands of a marine fish, the bonito (Katsuwonus plelamis), and characterized by amino acid sequence analysis in order to obtain additional evidence for duality of teleost GTHs. Glycoproteins were extracted from the pituitary glands, and intact GTHI and GTHII, consisting of two distinct subunits, were purified by ion-exchange chromatography on DEAE-cellulose, rpHPLC an Asahipak C4P-50 in alkaline buffer, and gel filtration on Superdex 75. The association of the subunits was stable in GTHI (39 kD) and unstable in GTHII (30 kD) in acidic conditions. Immunoblotting revealed that antisera against β subunits of chum salmon GTHs reacted with GTHII, but not with GTHI. In addition, none of the GTHs was stained with antiserum against human TSHβ. Sequence analysis demonstrated that bonito GTHIβ is homologous to salmon GTHIβ with 43% sequence identity, and bonito GTHIIβis homologous to salmon GTHIIβ with 67% identity. Sequence identity between bonito GTHIβ and GTHIIβ was only 28%. Thus, it is evident that the bonito pituitary gland produces two chemically distinct gonadotropins homologous to chum salmon GTHs.