Purification and characterization of lutropin receptor from membranes of pig follicular fluid.
Membranes derived from free floating granulosa cells in porcine ovarian follicular fluid were used as a starting material for structural characterization of both LH/hCG and FSH receptors. The receptors were highly hormone-specific and showed single classes of high-affinity binding sites (Kd = 19-74 pM). Their molecular weights as determined by affinity cross-linking with their respective 125I-ligands were similarly 70,000. The membrane-localized receptors could be solubilized with reduced Triton X-100 in the presence of 20% glycerol with good retention of hormone binding activity. The Triton extracts of membranes also showed hormone specificity and equilibrium binding constants similar to the membrane receptors (Kd = 32-48 pM). Affinity chromatography on divinylsulfonyl-Sepharose-oLH columns was utilized to purify the solubilized LH/hCG receptor to a specific activity of 2000 pmol/mg of protein. The purified receptor exhibited a high specificity for hCG and hLH but not for hFSH nor bTSH. The purified receptor was iodinated and visualized to be composed of a major protein of Mr approximately 70,000 and other minor proteins of molecular weights ranging from 14,000 to 40,000. Except for the Mr 14,000 protein, all other protein species bound to the concanavalin A-Sepharose column. The data suggest that the ovarian LH/hCG and FSH receptors are structurally similar and consist of a single polypeptide chain, as recently documented for the LH/hCG receptor (Loosefelt et al., 1989; McFarland et al., 1989).
Yarney
T A
TA
Reproduction Research Laboratory, Clinical Research Institute of Montreal, Quebec, Canada.
Sairam
M R
MR
Bhargavi
G N
GN
Mohapatra
S K
SK
D000818:Animals; D001667:Binding, Competitive; D002462:Cell Membrane / Q000032:analysis; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents / Q000378:metabolism; D005260:Female; D015571:Follicular Fluid / Q000032:analysis*; D011962:Receptors, FSH / Q000378:metabolism; D011967:Receptors, Gonadotropin / Q000378:metabolism; D011974:Receptors, LH / Q000032:analysis*; D012995:Solubility; D013552:Swine
D003432:Cross-Linking Reagents; D011962:Receptors, FSH; D011967:Receptors, Gonadotropin; D011974:Receptors, LH