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 bas s GtH II (stbGtH II), and its alpha and beta subunits were prepared fr om pituitaries of sexually mature hybrid striped bass. Pituitary glyco proteins were extracted with ethanol and intact stbGtH II purified by gel-filtration chromatography on Sephadex G-100, ion-exchange chromato graphy (IEC) on DE-52, and fast-performance liquid chromatography (FPL C) on Superdex 75. The presence of GtHs during the purification proced ure was monitored by characteristic elution on reversed-phase highperf ormance liquid chromatography (rpHPLC) and in vitro steroidogenic acti vity. The stbGtH II alpha and beta subunits were purified from the pit uitary ethanol extract by gel-filtration, IEC, and rpHPLC, and their i dentities assessed by sodium dodecyl sulfate-polyacrylamide gel electr ophoresis (SDS-PAGE), rpHPLC, and N-terminal amino acid sequencing. Mo lecular weights of intact stbGtH II and its alpha and beta subunits, d etermined by SDS-PAGE, were 34.5, 14.8, and 20.4 kDa, respectively. Th e stbGtH II beta subunit was used to produce specific antibodies, and a competitive enzyme-linked immunosorbent assay was developed using in tact stbGtH II for the standard curve. The sensitivity of the assay wa s 156 pg/ml (15.6 pg/well) and the intra-and interassay coefficients o f variation (at 50% binding) were 7.7% (n = 16) and 8.7% (n = 10), res pectively. Physiological validation of the assay was performed by meas uring changes of plasma GtH II levels in mature striped bass females, after injection of GnRHa ([D-Ala(6),Pro(9)-NEt]-mGnRH, 100 mu g/kg BW) . A maximum surge of GtH II in plasma was observed at 12 hr postinject ion (22.5 +/- 3.01 ng/ml), whereas GtH II levels in control fish (arou nd 4 ng/ml) remained unchanged. Displacement curves obtained with seri al dilutions of plasma and pituitaries from a number of perciform spec ies were parallel to the standard curve, indicating that this assay ca n be used for GtH II measurements in a variety of fish species. (C) 19 97 Academic Press.