Fig. 4-27. Representative two-ligand competition "free receptor" plot used to determine the equilibrium
dissociation constant (Kg-) for the binding of unlabeled corticosterone (B) to glucocorticoid
receptors labeled with [aH]dexamethasone in perfused mouse brain cytosol.
The points were derived from the data displayed in the Scatchard coordinate system in the left inset. The
nomenclature of the figure conforms to that of chapter III, with the identification of L (labeled ligand)
with [ H]dexamethasone and C (competing ligand) with corticosterone (B). The plot is described in the
text and in detail in chapter III. The abscissa was calculated by equation (4-9); equation (4-9) was also
used, in conjunction with the known corticosterone concentration (Sq), to find the ordinate, which is the
bound/free ratio for the competing corticosterone. The line (equation 4-11) was fit by simple linear
regression and was not constrained to intersect the orgin. The reciprocal of its slope is the equilibrium
dissociation constant (K^. or K^) for corticosterone: K^. = 5.2 nM. Right inset: the abscissa of the
plot described above has been changed from total free receptor to total bound receptor concentration in
order to display the range of receptor occupancy (0) spanned by the experimental conditions; range of 9:
67% to 96%. Left inset: Representative experimental data (generated by the "Edsall-Wyman" experimental
design and displayed in the Scatchard coordinate system) used to determine the dissociation constant for
corticosterone (Kg^) by competition with labeled [ Hjdexamethasone for glucocorticoid receptors. The
upper isotherm (-B,) was generated as described in the text and in the legend to figure 4-7, and the
lower curve (+8 nM B,A) was generated in an identical fashion with the same pool of cytosol, except that
both members of each pair of tubes contained 8 nM unlabeled corticosterone to reduce the slope of the
"Scatchard" plot as depicted. The lines were fit by simple linear regression, and the [ Hjdexamethasone
dissociation constant KgL and receptor concentration Bq were derived from the slope and intercept of the
upper [3H]dexamethasone Scatchard plot (#). These values of Kg^ and Bq were used, in conjunction with the
reduced slope of the lower plot (A) 5 as input to equation (4-8) to calculate the Kg. for corticosterone
(see text and also chapter III). The data (A were also transformed as described above into the
competition "free receptor" plot displayed in the figure. For the specific experiment displayed:
KgL = 2.3 nM, Bq = 2.5 nM (270 fmole/mg protein); the protein concentration was 9.3 mg/ml, and the
corticosterone = 5.9 nM (by use of the Edsali-Wyman equation).