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Gonadotropin binding factor(s). Extraction of high affinity gonadotropin binding sites from rat testis and partial characterization of their interaction with human follitropin, lutropin, and choriogonadotropin.

1976; Elsevier BV; Volume: 251; Issue: 16 Linguagem: Inglês

10.1016/s0021-9258(17)33206-4

1083-351X

Varun Bhalla, Joyce F. Haskell, H. J. Grier, V. Mahesh,

Reproductive Biology and Fertility

Factor(s) that bind gonadotropins have been extracted from rat testis by 30% ethanol (v/v) in water and their interaction with human lutropin (hLH) and human follitropin (hFSH) have been investigated by a new assay using dextran-coated charcoal.These studies reveal that:1. Maximal binding of gonadotropin with soluble factors was observed over a broad range of pH from 6.0 to 8.0 with a relative decline in binding at extremes of pH.The binding was independent of the ionic strength of the buffer and reached equilibrium within 5 min at 4", 27", and 37". 2. The soluble factors have marked thermostability, a point of distinction from detergent-solubilized receptors.3. The equilibrium dissociation constant (K,) of '%hFSH binding to the soluble factor was 6.0 * 0.58 x lo-l0 M, consistent with the values obtained from the membrane binding studies.Similarly, the Kd value for '*4-hLH to the soluble factor(s) was 3.33 l 0.3 x 10m9 M, comparable to the values obtained from the membrane binding studies.Hill plots demonstrated a lack of a cooperative relationship with an apparent Hill coefficient of 1.071 for hLH and 0.909 for hFSH.Furthermore, two classes of binding sites for '*51-human choriogonadotropin (hCG) were clearly discernible by both Lineweaver-Burk and Hill plots with an equilibrium dissociation constant of 2.4 + 0.5 x lo-" M and 1.35 * 1.2 x lo-' M. The apparent Hill coefficient of interaction of 'Y-hCG with the soluble factors was found to be 0.923 for high affinity and 1.09 for low affinity binding sites.4. The binding of '%hLH and **"I-hFSH with respect to concentrations of soluble factor(s) was found to be a saturable process, yielding an expected 4.4-fold higher K, for hLH (294 * 13.8 pg/ml) compared to hFSH (66.6 + 4 rg/ml).These findings are comparable with the equilibrium dissociation constants, thus confirming a 5-fold higher affinity of hFSH as compared to hLH for the soluble factors, i.e. the ratio of 3.0 x lo-' M to 6.0 x lo-l0 M versus the ratio of 294 pg/ml to 66.6 pg/ml.5. The hormone specificity of the interaction has been studied by using radiolabeled hFSH, hLH, hCG, prolactin, growth hormone, and bovine serum albumin.The binding of FSH at low factor concentrations was found to be 5. to lo-fold greater than prolactin, growth hormone, and albumin.6.The soluble factors are found in higher concentration in testis compared to liver, kidney, and blood.7. The effect of ethanol upon solubilization of the factor(s) has been investigated.The factor(s) can be extracted with buffer or water alone.However, 10 to 25%. of ethanol (v/v) facilitates the process of solubilization.The treatment with 70% ethanol (v/v) or more did not extract any factor activity from testes.The factor(s) were insoluble in petroleum ether, chloroform, absolute ethanol, methanol, or lipid solvent.8. Finally the effect of soluble factors on classical membrane binding was investigated.Increasing concentrations of the soluble factors resulted in graded inhibition of specific binding of '2JI-hLH and ""I-hFSH to fresh testicular homogenates.These studies implicate a possible regulatory role of soluble factors in hLH and hFSH activation of testis, more particularly as inhibitors of gonadotropin binding to testicular receptors.