Covalent cross-linking of radiolabeled human chorionic gonadotropin to rat ovarian luteinizing hormone receptor with glutaraldehyde.
Crude plasma membranes of pseudopregnant rat ovaries were incubated with 125I-labeled human chorionic gonadotropin (125I-hCG) and the formed luteinizing hormone (LH)/hCG receptor-125I-hCG complex was solubilized, partially purified by Sepharose 6B gel filtration, cross-linked with glutaraldehyde and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. An apparent molecular weight (mol wt) of 130,000 was obtained for the non-reduced complex. A similar-sized complex was observed, when 3H-hCG (beta-subunit labeled) instead of 125I-hCG (alpha-subunit labeled) was used, indicating that the complex contains intact hCG. Upon reduction of the cross-linked receptor-125I-hCG complex, a 105,000 mol wt complex in addition to the 130,000 one was observed. No smaller complexes appeared, however, upon reduction of the receptor-3H-hCG complex, suggesting that the alpha-subunit of hCG predominantly interacts with the receptor. The cross-linking efficiency was dependent on protein concentration, glutaraldehyde concentration, pH, reaction time and temperature. Under optimal conditions (2 mM glutaraldehyde, pH 7.0-8.0, 60 min, 20 degrees C) no nonspecific complexes appeared and the efficiency of cross-linking of the partially purified detergent-solubilized receptor-125I-hCG complex was approximately 30%. Glutaraldehyde thus provides a rapid and efficient cross-linking reagent to covalently attach 125I-hCG to its receptor and is likely to be highly applicable to other receptor-ligand systems as well.
D000447:Aldehydes / Q000494:pharmacology*; D000818:Animals; D006063:Chorionic Gonadotropin / Q000302:isolation & purification*; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005976:Glutaral / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D010053:Ovary / Q000032:analysis*; D051381:Rats; D011919:Rats, Inbred Strains; D011974:Receptors, LH / Q000302:isolation & purification*; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013696:Temperature
D000447:Aldehydes; D006063:Chorionic Gonadotropin; D003432:Cross-Linking Reagents; D011974:Receptors, LH; D005976:Glutaral