Solubilization and purification of the lutropin (LH) receptor from porcine testes.

Abstract

The LH receptor was solubilized from porcine testis homogenates by different detergents. Non-ionic detergents appeared to be the best ones regarding solubilization yield and recovery of active receptor after elution from hCG affinity gels. However, the final yield was not greater than 1-2% of the starting receptor activity. We also investigated the effect of added phospholipids (75% phosphatidylcholine + 25% phosphatidylethanolamine) on the yield of the overall purification process. It was shown that the best p value [i.e. the ratio of (detergent concentration - critical micellar concentration) to phospholipids] upon solubilization was congruent to 1.1 for a non-ionic detergent such as Nonidet P-40, while a higher p value was better upon elution. The stability of the solubilized receptor versus pH and SDS has been studied. The receptor exhibited two pKa's of denaturation congruent to 3.8 and 11.1, while the [125I]hCG-receptor complex dissociated with a pKa congruent to 3.8 and 10.3 SDS concentrations as low as 0.05% denatured rapidly and apparently, irreversibly, the solubilized LH receptor. The stability of different affinity gels was checked and it was found that the best yield for receptor elution (congruent to 10%) was obtained with an immunoaffinity anti-hCG support. The obtention of two monoclonal antibodies is mentioned, as well as their competition with the binding of [125I]pLH to testis homogenates.