Photoaffinity labeling of human chorionic gonadotropin-binding sites in rat ovarian plasma membranes.
A photoaffinity derivative of human chorionic gonadotropin (hCG-N-hydroxysuccinimidyl-4-azidobenzoate (hCG-HSAB) ) was used to identify components of the lutropin (LH)/hCG receptor in plasma membranes prepared from pregnant mare serum-pretreated rat ovaries. In intact plasma membranes, the Kd for hCG (1 X 10(-10) M) was the same with both 125I-hCG and 125I-hCG-HSAB, and the binding capacity for the photoaffinity label (1 pmol/mg of membrane protein) was the same as for native hCG. Sodium dodecyl sulfate-gel electrophoresis under reducing conditions of 125I-hCG-HSAB cross-linked to plasma membranes revealed newly formed complexes of Mr = 106,000, 85,000, and 80,000 (representing membrane protein components of Mr = 86,000, 65,000, and 60,000, respectively). However, only the Mr = 106,000 complex displayed an affinity for hCG (Kd of 1 X 10(-10) M) consistent with the physiological LH/hCG receptor. The other binding sites were nonsaturable. Half-maximal saturation by 125I-hCG-HSAB of the Mr = 106,000 complex (1-2 X 10(-9) M) was similar to the Kd of the specific binding of 125I-hCG-HSAB to intact plasma membranes (8 X 10(-10) M). The present data suggest that among the different components of the LH/hCG receptor, only one component (of Mr = 86,000) possesses the affinity and binding capacity characteristics of the LH/hCG receptor as determined using radiolabeled, underivatized hCG.
D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001386:Azides / Q000138:chemical synthesis / Q000378:metabolism*; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D006063:Chorionic Gonadotropin / Q000378:metabolism*; D005260:Female; D007700:Kinetics; D008970:Molecular Weight; D010053:Ovary / Q000378:metabolism*; D010777:Photochemistry; D051381:Rats; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D011974:Receptors, LH
D000345:Affinity Labels; D001386:Azides; D006063:Chorionic Gonadotropin; D011956:Receptors, Cell Surface; D011974:Receptors, LH; C029534:hydroxysuccinimidyl-4-azidobenzoate