Specific binding of [³H]17α,20β-dihydroxy-4-pregnen-3-one to oocyte cortices of rainbow trout (Oncorhynchus mykiss)

Specific binding of [³H]17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DP) to plasma membranes prepared from defolliculated oocytes of rainbow trout (Onchorhynchus mykiss) was identified and characterized. Binding was rapid and reached equilibrium in 30 min. 17α,20β-DP strongly inhibited [³H] 17α,20β-DP binding in a competitive manner. Scatchard analysis revealed two different binding sites: a high affinity binding site with a K_d of 18 nM and a B_max of 0.2 pmoles/mg protein; and a low affinity binding site with a K_d of 0.5 μM and a B_max of 1 pmoles/mg protein. This binding activity was successfully solubilized with n-heptyl-β-D-thioglucoside. [³H]17α,20β-DP binding to solubilized preparations reached equilibrium in 1h, and was competitively inhibited with 17α,20β-DP and 17α,20β,21-trihydroxy-4-pregnen-3-one. However, Scatchard analysis showed a single binding site with a K_d of 0.3 μM. The reason for the disappearance of the high affinity binding site in solubilized preparations remains unclear. These results demonstrate that a specific binding site for 17α,20β-DP exists in the plasma membrane of rainbow trout oocytes.