Page 264 - The Toxicology of Fishes

244 The Toxicology of Fishes

Specific Binding (fmol/mg) 60 Scatchard
A 80
70
50
40
30
Langmuir
20
10
0
4
0 1 2 3 Simultaneous 5
Free TCDD (nM)
B 35
Specific Binding (fmol/mg) 25 Scatchard
30

20
15
Langmuir
10
5
0
0 0.5 1 1.5 2 Simultaneous 3
2.5
Free TCDD (nM)

FIGURE 5.6 Effect of data quality on curve fits. Specific binding of [ H]-TCDD was measured in PLHC-1 fish hepatoma
3
cells (A) and CDK dolphin kidney cells (B), and the data were fit using the Scatchard method (Equation 5.7), directly fit
to the Langmuir binding isotherm (Equation 5.6), or by simultaneous fitting of the total and nonspecific binding data
(Equations 5.9 and 5.10). Binding curves predicted using the constants determined from the fits (Table 5.2) are plotted. The
CDK cells were generously provided by Dr. Michael Carvan, and the data are shown with his permission.

yielded quite different estimates of K and R for PLHC-1 cells but nearly identical estimates for CDK
D
T
cells (Table 5.2). Notice that as the theoretical appropriateness of the fits increases (moving down the
table), the K and R decrease. Overestimation of the R is common when a few points with higher specific
T
D
T
binding skew the fit, as the two points near 70 fmol/mg of specific binding do for the PLHC-1 data.
Efficacy
Interaction with a ligand is only the first step in the physiological role of the receptor. Ligand binding
leads to a change in protein conformation that may activate the receptor (often referred to as receptor
activation or receptor transformation). Depending on the specific receptor involved, activation may
involve enzyme (e.g., protein kinase) or transport (e.g., ion channel) activity by the receptor protein
itself, interaction with DNA response elements, or (most frequently) interaction with other proteins that
help transduce the receptor signal. The structure of the ligand–receptor complex will vary depending on

TABLE 5.2
Binding Constants Predicted Using Different Fit Models
Fit Model K D (nM) R T a
PLHC-1 Scatchard 1.22 84
Langmuir 0.95 77
Simultaneous 0.50 53
CDK Scatchard 0.57 37.6
Langmuir 0.58 37.5
Simultaneous 0.57 37.1
3
a Expressed as fmol [ H]-TCDD bound per mg cell protein.