JBC, Vol. 251, Issue 16, 4947-4957, Aug, 1976
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
V. K. Bhalla, J. Haskell, H. Grier and V. B. Mahesh
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 degrees, 27 degrees, and 37 degrees. 2. The
soluble factors have marked thermostability, a point of distinction from
detergent-solubilized receptors. 3. The equilibrium dissociation constant
(Kd) of 125I-hFSH binding to the soluble factor was 6.0 +/- 0.58 X 10(-10)
M, consistent with the values obtained from the membrane binding studies.
Similarly, the Kd value for 125I-hLH to the soluble factor(s) was 3.33 +/-
0.3 X 10(-9) 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 125I-human choriogonadotropin
(hCG) were clearly discernible by both Lineweaver-Burk and Hill plots with
an equilibrium dissociation constant of 2.4 +/- 0.5 X 10(-11) M and 1.35
+/- 1.2 X 10(-9) M. The apparent Hill coefficient of interaction of
125I-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 125I-hLH and
125I-hFSH with respect to concentrations of soluble factor(s) was found to
be a saturable process, yielding an expected 4.4-fold higher Kd for hLH
(294 +/- 13.8 mug/ml) compared to hFSH (66.6 +/- 4 mug/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 10(-9) M to 6.0 X 10(-10) M versus the
ratio of 294 mug/ml to 66.6 mug/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 10-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...
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