Solubilization and characterization of high affinity follicle-stimulating hormone receptors from porcine granulosa cells.
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 x 10(10) M-1 to 8 x 10(10) M-1 in 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.
Department of Obstetrics and Gynecology, University of Cincinnati College of Medicine, OH 45267-0526.
S J
SJ
Sperbeck
D Y
DY
Song
A R
AR
LaBarbera
D000818:Animals; D005260:Female; D005640:Follicle Stimulating Hormone / Q000378:metabolism; D006107:Granulosa Cells / Q000737:chemistry*; D008970:Molecular Weight; D011962:Receptors, FSH / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility; D013552:Swine; D013696:Temperature
D011962:Receptors, FSH; D005640:Follicle Stimulating Hormone