Solubilization and Characterization of High Affinity Follicle-Stimulating Hormone Receptors from Porcine Granulosa Cells

Abstract

Follicle-stimulating hormone (FSH)-receptors were solubilized from immature porcine ovarian granulosa cells with retention of high affinity ¹²⁵I-porcine FSH-binding activity. The optimal concentration of Triton X-100 for solubilization was 0.5% (w/v), and the optimal cellular protein concentration 25 mg/ml. Glycerol (30%) increased recovery of solubilized receptor. ¹²⁵I-pFSH-binding affinity ranged from 4 × 10¹⁰ M⁻¹ to 8 × 10¹⁰ M⁻¹ inn either the absence or presence of glycerol. ¹²⁵I-pFSH-binding capacity was 5 fmol/mg protein in the absence of glycerol and 58 fmol/mg protein in the presence of glycerol as determined by equilibrium saturation binding analysis. By gel permeation chromatography, the apparent size of the ¹²⁵I-pFSH-receptor complex was 462 kDa in the absence of glycerol and 762 kDa in the presence of glycerol. Ligand blotting of solubilized receptor yielded a single species with an apparent molecular weight of 200 kDa under nonreducing conditions and a single species with an apparent molecular weight of 60 kDa under reducing conditions. These studies indicated that high affinity FSH-binding activity can be solubilized from membranes of immature porcine granulosa.