PARTIAL CHARACTERIZATION OF A SEX-STEROID BINDING-PROTEIN IN THE SPOTTED SEA-TROUT (CYNOSCION NEBULOSUS)

A sex-steroid binding protein (SBP) that binds both testosterone and estradiol-17 beta was identified in the plasma and ovarian interstitial fluids of the spotted seatrout, Cynoscion nebulosus. Scatchard analysis revealed a single binding component with dissociation constants of 4.89 +/- 0.19 and 3.13 +/- 0.11 nM and binding capacities of 423 +/- 25 and 482 +/- 24 nM for testosterone and 17 beta-estradiol respectively (n = 5). Competition studies demonstrated that the seatrout SBP had a high affinity for a range of estrogens and androgens; much lower affinity for 11-ketotestosterone, progesterone, and pregnenolone; and even lower affinity for cortisol and the maturation-inducing steroid, 17 alpha,20 beta,21-trihydroxy-4-pregnen-3-one. The rates of steroid association and dissociation were very rapid with a t(1/2) of less than 30 sec for ligand association and 30 sec for ligand dissociation. Testosterone binding activity precipitated between 40 and 60% ammonium sulphate saturation and eluted as a single peak with an estimated molecular mass of 150 kDa on Sephacryl S-200. Electrophoresis of plasma on native PAGE over a range of acrylamide concentrations gave a molecular mass estimate of 135 kDa The migration of 17 beta-estradiol binding activity on native PAGE was identical to that of the testosterone binding activity. Steroid binding activity was destroyed by exposure to EDTA or dithiothreitol (DIT), or to temperatures above 50 degrees C. The SBP found in the ovarian interstitial fluids was similar to the plasma binding protein in terms of saturability, affinity, specificity, association and dissociation races, stability, and molecular weight.