3
Fig. 4-17. Merged nonlinear regression analysis of the binding of [ H]corticosterone to
the complete ensemble of corticosterone binding sites in perfused mouse brain
cytosol (derived from the values of total binding, By, from 7 independent
experiments combined with the individual estimates, for each experiment, of the
receptor concentration, BM.Y, derived from initial Scatchard plots such as that
depicted in figure 4-12).
The data and the resulting regression have been plotted in a "Scatchard-1ike" coordinate
system (where Bgp or By has been replaced by By/B^, the normalized value of total bound
steroid). Representative experimental conditions are indicated in the legend to
figure 4-8, and the use of the nonlinear regression program is described in Methods. The
model to which the data were fit consists of the sum of one saturable high-affinity
(specific) component and one nonsaturable (nonspecific) component as described by
equation (4-6). The merged regression equation does not contain F; it accepts By, BMAX
(substituted for Bq), and (total [^H]corticosterone concentration) as sets of input
variables and then regresses By on the variables and B^x, fitting the adjustable
binding parameters Kd and (the asymptotic "sink" of nonspecific binding). Each input
data point was weighted as 1/By^, consistent with the assumption that the coefficient of
variation or percentage error in the measurement of By is constant over the range of By.
For display the resulting complete 2-parameter regression model was plotted in the
"Scatchard-1ike" coordinate system (curved line), and each component was also plotted
separately (2 straight lines). (Note that the x-axis is plotted below y=0 in the figure.)
Equilibrium dissociation constant K. = 6.6 0.3 nM (n = 7 experiments, 50 points).
Cl ^
Cj = 0.88 x 10 M Range of total binding site concentration 4.1 7.5 nM; range
of cytosol protein concentration 4.8 9.3 mg/ml.