Characterization of Testicular GH Binding Sites
The specific binding of 125I-rtGH to increasing amounts of crude testicular preparations, obtained at two different stages of spermatogenesis (stages I and VI), was dependent on membrane concentration in the range of 5 to 40-50 mg of pellet per tube (~0.5 to 4-5 mg of protein per tube). In the subsequent experiments, we used 20 mg of pellet per tube for quantitative studies (single-point binding) and 20 mg (stages I-IV) or 50 mg (stages V-VIII) of pellet per tube for saturation experiments.
Binding specific to 125I-rtGH appeared saturable when increasing concentrations of 125I-rtGH were incubated with a fixed amount of testis (Fig. la). Scatchard plot analyses were always linear, indicating the presence of a single population of binding sites with high affinity (Ka = 4.5 X 109 ± 0.25 M1, n = 21), as well as a limited number of sites. The affinity constants in crude testis preparations were of the same order of magnitude as those observed in testis membrane preparations (Fig. lb) and crude liver preparations (Fig. lc). The specific activity (fmol/mg protein) of the crude testis preparation was 3- to 6-fold lower (depending on the stage) than in the testis membrane preparation. buy prednisone
FIG. 1. Effect of increasing concentrations of [l25l]rtCH (20 000-1 000 000 cpm/tube) on specific binding to a) crude testis preparation (50 mg of pellet per tube), b) testis membrane preparation (50 mg of pellet per tube), and c) crude liver preparation (5 mg of pellet per tube). The inserts represent the derived Scatchard plots. Scatchard plot analysis were performed with values for free (U) hormone corrected for MBA of the tracer.