0 100 200 300 400 B(fmoles/ml)

5000 10,000

0 100 200 300 400 B(fmoles/ml)

5000 10,000

FIGURE 1-32 Scatchard analysis of curves representing two binding components. Curve 1: Scatchard plots of total binding. Curve 2: Linear extrapolation of the high-affinity component that includes contribution from the low-affinity component. Curve 3: Specific binding of the high-affinity component after removal of the nonspecific contribution, representing the true high-affinity curve. [Redrawn from Chamness, G. C. and McGuire, W. L. (1975). Scatchard plots: Common errors in correction and interpretation. Steroids 26,538.

1. Autoradiogoraphic Localization of Hormones

In a typical experiment, a physiological dose of radioactive hormone, usually labeled with either tritium or carbon-14, is administered to an animal. At the appropriate time, the tissues of interest are collected, fixed, sectioned, and exposed to film for extensive intervals of time. Figures 1-28C and 1-28D displays the results of administration of tritiated estrogen to MCF-7 human breast cancer cells; the results clearly indicate that the steroid hormone-receptor complex is associated with the nucleus of the cell. In Figures 1-28A and 1-28B, the localization of the unoccupied and occupied estrogen receptors was studied through the application of fluorescent monoclonal antibodies to the estrogen receptor. It is apparent that both the unoccupied and occupied forms of the estrogen receptor are found in the nucleus of the target cell.

2. Saturation Analysis

A very informative technique for studying the interaction of radioactive preparations of hormone with homogenates of target cells (which contain highly impure receptor) or with partially purified or purified receptor preparations is the procedure of saturation analysis. Since the cognate receptor has a highly specific ligand-binding domain, under usual incubation conditions the noncovalent hormone-receptor complex is formed rapidly. The interaction of the hormone (H) and the unoccupied receptor (R) to form receptor-hormone complex (RH) can be formally described in the following mathematical fashion:

The association of H and R to yield HR and the dissociation of HR into H and R are readily reversible processes, i.e., it is a dynamic equilibrium since the hormone does not become covalently bound to the receptor. Thus, the equilibrium can be expressed in terms of the association constant, K^ which is mathematically equivalent to 1 / dissociation constant (Kd):

The individual rate constants fc+1 and numerically describe the rates of the forward (on-rate) and backward (off-rate) reactions, respectively, as written in Eq. (1). Experimentally, the approach to equilibrium can be followed by a progress curve of binding that reaches saturation (see Figure 1-29). A saturating amount of hormone is determined by using variable amounts of

0 0

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