Characterization of a Follitropin-binding Component Prepared from Immature Bovine Testes in the Absence of Detergent*

Buffer (0.05 M Tris, pH 7.5) soluble factors capable of specifically binding radioiodinated human follitropin ('251-hF!3H) were detected in high speed supernatants (cytosol) of calf testes homogenates. This '2SI-hFSH binding activity, designated as the buffer-soluble binding component (BSBC), did not sediment at 250,000 X g, passed a 0.45 p filter, and was equally distributed throughout the tissue supernatant solution after ultracentrifugation. No vesicular structures could be detected upon electron microscopy. BSBC activity was present in similarly prepared cytosol fractions from rat testis, but not bovine or rat liver, spleen, or kidney. The binding of '261-hFSH to BSBC from calf testis could be specifically inhibited by small quantities of unlabeled hFSH in a dose-related fashion (12 ng of hFSH = 50% binding inhibition), at a level of sensitivity comparable to that previously reported for systems using calftestes membrane or detergent-solubilized membrane receptor. The '261-hFSH-binding capacity of calf testes BSBC represented 39% of the total capacity of testis to bind radioligand. However, only 20% of '2sI-hFSH-binding capacity of 31,000 X g testes membrane fraction could be solubilized by buffer upon homogenization. Thus, although a portion of BSBC activity could be derived from membranes, the greater portion of binding activity seems to be from some other source, presumably the cytoplasm. Binding of '261-hFSH to BSBC was saturable when increasing concentrations of radioligand were added to a constant amount of BSBC protein and when a constant amount of radioligand was added to increasing amounts of BSBC protein. The K, of the BSBC was 2.9 X lo9 M" with a binding capacity of 2 X 10-l~ mol/mg of protein. These values were not significantly different from those obtained using calf testes membrane or detergent-solubilized membrane receptor systems. When BSBC from calf testis was filtered through a column of Sephacryl S-300 (0.05 M Tris, pH 7.6, 4 "C) in the presence of 0.1% Triton X-100, "'IhFSH-binding activity eluted in a region corresponding to a molecular weight of about 400,000. When filtered through the same column in the absence of detergent, FSH-binding activity was detected in the column void volume indicating a molecular weight in excess of 1 X 10' and suggesting aggregation may have occurred in the absence of detergent. When preformed 1261-hFSHBSBC complex was filtered through the same column and in the presence of detergent, radioactivity eluted predominately in a position corresponding to a molec-