Purification and characterization of follicle-stimulating hormone from pituitary glands of sea bass (Dicentrarchus labrax).
Follicle-stimulating hormone (FSH) was purified from pituitaries of sea bass (Dicentrarchus labrax), and its biochemical and biological properties were studied. Sea bass FSH (sbsFSH) was purified by ethanol extraction-precipitation (40-85%), followed by anion-exchange chromatography on a LKB Ultropac TSK-DEAE column using a linear gradient of ammonium bicarbonate (50-1000 mM) and reverse phase chromatography on a RESOURCE 15RPC column with a linear gradient of acetonitrile (0-50%), using a FPLC system. The molecular mass of the purified sbsFSH, estimated by mass spectrometry, was of 28.5 kDa for the dimer, 12.6 kDa for the glycoprotein alpha (GPalpha) and 13.6 kDa for FSHbeta subunits. After separation by SDS-PAGE under reducing condition, the intact sbsFSH was dissociated in the respective subunits (GPalpha and FSHbeta). Subunit identity was confirmed by immunological detection and N-terminal amino acid sequencing. Deglycosylation treatment with N-glycosidase F, decreased the molecular mass of both subunits. Intact sbsFSH activated the sea bass FSH receptor stably expressed in the cell line HEK 293, in a dose dependent manner. Purified sbsFSH showed gonadotropic activity, by stimulating the release of estradiol-17beta (E2) from sea bass ovary and testosterone (T) and 11-ketotestosterone (11KT) from testicular tissue cultured in vitro, in a dose and time dependent manner. These results showed that the purified sbsFSH is a heterodimeric hormone, composed of two distinct glycoprotein subunits (GPalpha and FSHbeta), and has biological activity judged by its ability to stimulate its receptor in a specific manner and to promote steroid release from gonadal tissue fragments.
Department of Fish Physiology and Biotechnology, Instituto de Acuicultura de Torre la Sal, Consejo Superior de Investigaciones Científicas, 12595 Torre la Sal, Ribera de Cabanes s/n, Castellón, Spain.
Gregorio
G
Molés
Ana
A
Gómez
Ana
A
Rocha
Manuel
M
Carrillo
Silvia
S
Zanuy
D000595:Amino Acid Sequence; D000818:Animals; D001492:Bass / Q000378:metabolism; D005260:Female; D005640:Follicle Stimulating Hormone / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism / Q000502:physiology; D008297:Male; D008969:Molecular Sequence Data; D010902:Pituitary Gland / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011962:Receptors, FSH / Q000032:analysis / Q000378:metabolism; D011974:Receptors, LH / Q000032:analysis / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D021122:Protein Subunits; D011962:Receptors, FSH; D011974:Receptors, LH; D005640:Follicle Stimulating Hormone