Solubilization and some characteristics of the follitropin receptor from calf testis.

Immature calf testes were found to be an unusually rich source of follitropin (FSH) receptors. Particulate fractions derived from such testes bound 32% of added biologically active radiolabeled human FSH (12”I-labeled hFSH) and had a binding capacity of 52 x lo-l4 mol/mg of protein. These values are considerably higher than those previously reported for FSH receptors in other types of beef or mature and immature rat testis. Solubilization of the receptor was achieved by extraction with Triton X-100. Its presence in detergent extracts of immature calf testes was demonstrated by gel filtration and sucrose density gradient centrifugation experiments wherein the position of 12”I-labeled hFSH.soluble receptor complex was determined after incubation of radioligand with soluble receptor in the absence and presence of lOOO-fold excess unlabeled hormone. Separation of free ‘““I-labeled hFSH from that bound to soluble receptor was done by double precipitation with polyethylene glycol, with inclusion of appropriate controls to correct for co-precipitation of free 12”I-labeled hFSH with the hormone.receptor complex. Binding of the lZZI-labeled hFSH to soluble receptor was pHand temperature-dependent, being maximal at pH 7.5 and 24” after 3 h of incubation, and could be completely inhibited by excess unlabeled hormone. Large amounts (5000 ng) of other pituitary hormones did not inhibit binding of 12”I-labeled hFSH to soluble receptor beyond that attributable to trace contamination with native hFSH. The characteristics of the ‘2”I-labeled hFSH binding inhibition curve obtained with graded doses of unlabeled hormone were similar whether solubilized or particulate receptors were utilized. Triton X-100 extracts of particulate fractions from such nongonadal tissue as liver, kidney, and spleen did not result in solubilization of factors capable of binding W-labeled hFSH, demonstrating the tissue specificity of the Triton X100~solubilized testicular receptor. The binding capacity of solubilized receptor derived from immature calf testes, 19 X lo-l4 mollmg of protein, was significantly (64%) less than that seen with particulate receptor prior to detergent extraction, but at least as high as that seen for FSH particulate receptors in larger beef testis or testis from mature or immature rats. Various trinucleotides (1 mM) inhibited the binding of 1Z51-labeled hFSH and also promoted dissociation of specifically bound i2”I-labeled hFSH from preformed hormone. soluble receptor complex,