Purification of gonadotropin II from a teleost fish, the hybrid striped bass, and development of a specific enzyme-linked immunosorbent assay.
A highly purified gonadotropin II (GtH II), referred to as striped bass GtH II (stbGtH II), and its alpha and beta subunits were prepared from pituitaries of sexually mature hybrid striped bass. Pituitary glycoproteins were extracted with ethanol and intact stbGtH II purified by gel-filtration chromatography on Sephadex G-100, ion-exchange chromatography (IEC) on DE-52, and fast-performance liquid chromatography (FPLC) on Superdex 75. The presence of GtHs during the purification procedure was monitored by characteristic elution on reversed-phase high-performance liquid chromatography (rpHPLC) and in vitro steroidogenic activity. The stbGtH II alpha and beta subunits were purified from the pituitary ethanol extract by gel-filtration, IEC, and rpHPLC, and their identities assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), rpHPLC, and N-terminal amino acid sequencing. Molecular weights of intact stbGtH II and its alpha and beta subunits, determined by SDS-PAGE, were 34.5, 14.8, and 20.4 kDa, respectively. The stbGtH II beta subunit was used to produce specific antibodies, and a competitive enzyme-linked immunosorbent assay was developed using intact stbGtH II for the standard curve. The sensitivity of the assay was 156 pg/ml (15.6 pg/well) and the intra- and interassay coefficients of variation (at 50% binding) were 7.7% (n = 16) and 8.7% (n = 10), respectively. Physiological validation of the assay was performed by measuring changes of plasma GtH II levels in mature striped bass females, after injection of GnRHa ([d-Ala6,Pro9-NEt]-mGnRH, 100 microg/kg BW). A maximum surge of GtH II in plasma was observed at 12 hr postinjection (22.5 +/- 3. 01 ng/ml), whereas GtH II levels in control fish (around 4 ng/ml) remained unchanged. Displacement curves obtained with serial dilutions of plasma and pituitaries from a number of perciform species were parallel to the standard curve, indicating that this assay can be used for GtH II measurements in a variety of fish species.
Mañanós
E L
EL
Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, Maryland 21202, USA.
Swanson
P
P
Stubblefield
J
J
Zohar
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001492:Bass; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D007987:Gonadotropin-Releasing Hormone / Q000031:analogs & derivatives; D006065:Gonadotropins, Pituitary / Q000737:chemistry / Q000302:isolation & purification*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D010902:Pituitary Gland / Q000737:chemistry; D017421:Sequence Analysis
D006065:Gonadotropins, Pituitary; D010446:Peptide Fragments; C079737:gonadotropin II, Katsuwonus; D007987:Gonadotropin-Releasing Hormone