Isolation and characterization of FSH and LH from pituitary glands of Atlantic halibut (Hippoglossus hippoglossus L.).
Two gonadotropins (GtH), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), were isolated and characterized from pituitary glands of Atlantic halibut (Hippoglossus hippoglossus L.). Glycoproteins were extracted in 40% ethanol followed by precipitation in 85% ethanol. Subsequently, glycoproteins were fractionated by ion-exchange chromatography on a Whatman DE-52 column using a stepwise gradient of ammonium bicarbonate (50-1000 mM). Intact FSH and LH were finally purified on rpHPLC using an AsahiPak C4P-50 column with an acetonitrile gradient (10-60%). SDS-PAGE showed a molecular mass of 33 and 32 kDa for intact FSH and LH, respectively. Final purification of subunits was performed by a subsequent purification step on rpHPLC using a Phenomenex Jupiter C18 column with an acetonitrile gradient (10-60%). FSHbeta, LHbeta, and the common alpha subunit showed molecular masses of 25, 24, and 19 kDa, respectively. Subunit identity was confirmed by N-terminal amino acid sequencing. Intact FSH and LH showed gonadotropic activity by stimulating release of 11-ketotestosterone from turbot (Scophthalmus maximus L.) testicular tissue in vitro. This provides the first purification of two distinct GtHs from an evolutionary advanced pleuronectiform teleost.
Weltzien
Finn-Arne
FA
Institute of Marine Research, Austevoll Aquaculture Research Station, 5392 Storebø, Norway. f.a.weltzien@bio.uio.no
Norberg
Birgitta
B
Swanson
Penny
P
D000738:Androsterone / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D001681:Biological Assay; D005432:Flounder / Q000378:metabolism*; D005640:Follicle Stimulating Hormone / Q000302:isolation & purification* / Q000378:metabolism; D007986:Luteinizing Hormone / Q000302:isolation & purification* / Q000378:metabolism; D008297:Male; D010902:Pituitary Gland / Q000166:cytology / Q000378:metabolism*; D013737:Testis / Q000378:metabolism; D013739:Testosterone / Q000378:metabolism
C015961:11-ketoandrosterone; D013739:Testosterone; D007986:Luteinizing Hormone; D005640:Follicle Stimulating Hormone; D000738:Androsterone