The rat ovarian lutropin receptor. Purification, hormone binding properties, and subunit composition.
The luteinizing hormone/human choriogonadotropin (hCG) receptor from superovulated rat ovary was purified to homogeneity. A novel scheme based on reverse immunoaffinity chromatography using immobilized antibodies to membrane proteins from receptor down-regulated ovary and subsequent two-step affinity purification on hCG-Sepharose was used to isolate homogeneous receptor. The purification method was also compared to an alternate scheme involving lectin affinity chromatography followed by hCG affinity chromatography. The purified receptor obtained by the latter method was heterogeneous and highly aggregated. The hormone binding properties, molecular size, and subunit composition of the purified receptor obtained by either method were identical. The stability of the receptor during and following solubilization was markedly improved by using 20% glycerol. The pure receptor consists of four nonidentical subunits of molecular weight 79,300 (alpha), 66,400 (beta), 55,300 (gamma), and 46,700 (delta) as indicated by polyacrylamide gel electrophoresis under reducing conditions. All receptor subunits generally, but occasionally excepting the alpha-subunit, were specifically labeled with iodinated hCG in membrane and soluble receptor preparations using bifunctional cross-linking agents. Analysis of the cross-linked hormone-receptor complexes under nonreducing conditions showed the molecular mass of the undissociated receptor to be 268,000 daltons. Hormone binding studies demonstrated that the isolated receptor retained all of the specific binding characteristics expected for the luteinizing hormone/hCG receptor. In combination, these results indicate that the functional and structural properties of the receptor were not altered during purification.
D000818:Animals; D001667:Binding, Competitive; D002462:Cell Membrane / Q000032:analysis; D006063:Chorionic Gonadotropin / Q000378:metabolism; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007158:Immunologic Techniques; D007986:Luteinizing Hormone / Q000378:metabolism; D008970:Molecular Weight; D009439:Neuraminidase / Q000494:pharmacology; D010053:Ovary / Q000032:analysis*; D051381:Rats; D011919:Rats, Inbred Strains; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D011974:Receptors, LH
D006063:Chorionic Gonadotropin; D003432:Cross-Linking Reagents; D011956:Receptors, Cell Surface; D011974:Receptors, LH; D007986:Luteinizing Hormone; D009439:Neuraminidase