Purification, characterization, and amino-terminal sequence of rat ovarian receptor for luteinizing hormone/human choriogonadotropin.
The luteinizing hormone (LH)/human choriogonadotropin (hCG) receptor of rat ovary was solubilized with Lubrol PX in the presence of 20% glycerol and protease inhibitors, and purified by one-step affinity chromatography. Purified receptor had a specific hCG binding capacity of 4900 pmol/mg protein, and displayed a single class of high affinity binding sites (Ka = 6.20 X 10(9) M-1). An 11,200-fold purification over the starting crude homogenate was achieved. The purified LH/hCG receptor was identified by sodium dodecyl sulfate-gel electrophoresis and silver staining as a single protein of 92 kDa. The ability of the purified 92-kDa protein to specifically bind hormone was demonstrated by electroblotting onto Immobilon P membrane, incubation with 125I-labeled hCG, and autoradiography of the blot. In addition to a 92-kDa band, ligand blotting also yielded a 170-kDa band representing receptor dimer. Covalent cross-linking of hCG, with isotope in either the alpha- or beta-subunit, to membrane-bound receptor produced complexes that contained a single receptor component of approximately 92 kDa. The cross-linking studies indicated that both subunits interact with receptor and also suggested receptor dimer formation. Following sodium dodecyl sulfate-electrophoresis, purified receptor was electroblotted onto polyethylenimine-treated glass fiber filters for direct microsequencing in a gas-phase sequenator. Eleven cycles of sequence analysis yielded the unique sequence: NH2-Arg-Glu-Leu-Ser-Gly-Ser-Leu-XXX-Pro-Glu-Pro-COOH. These results indicate that the rat ovarian LH/hCG receptor is a protein of 92 kDa which can be easily purified in microgram amounts. This study also describes a relatively simple technique for electroblotting and microsequencing that should be applicable to other membrane-bound hormone receptors.
Roche
P C
PC
Department of Biochemistry and Molecular Biology, Mayo Graduate School of Medicine/Mayo Foundation, Rochester, Minnesota 55905.
Ryan
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D006063:Chorionic Gonadotropin / Q000378:metabolism; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007986:Luteinizing Hormone / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010053:Ovary / Q000737:chemistry*; D011145:Polyvinyls; D051381:Rats; D011974:Receptors, LH / Q000302:isolation & purification*; D005465:Fluorocarbon Polymers
D006063:Chorionic Gonadotropin; D003432:Cross-Linking Reagents; D007986:Luteinizing Hormone; D046911:Macromolecular Substances; D011145:Polyvinyls; D011974:Receptors, LH; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers