Comparison of binding parameters for [3H]OFQ and125I-Tyr14-OFQ in 293/OFQ-R and CHO/OFQ-R membranes
| 293 OFQ-R | CHO OFQ-R | |||
|---|---|---|---|---|
| [3H]OFQ | 125I-Tyr14-OFQ | 125I-Tyr14-OFQ | ||
| Kinetic parameters | ||||
| ka (mmin) (× 108) | 5.6 ± 1 (n = 4) | 14.6 ± 0.1 (n = 3) | 26.0 ± 0.3 (n = 3) | |
| k d1 (min−3) (× 10−3) | 3.2 ± 0.2 (n = 3) | 8.7 ± 0.6 (n = 2) | 3.6 ± 0.1 (n = 3) | |
| % Slow | 45% | 71% | ||
| k d2 (min−1) (× 10−3) | 91.5 ± 2.5 (n = 3) | 202 ± 1 (n = 2) | ||
| % Fast | 55% | 29% | ||
| KdH (pm) | 5.73 | 5.95 | 1.39 | |
| KdL (pm) | 165 | 138 | ||
| Equilibrium parameters | ||||
| KdH * (pm) | 8.4 ± 1.3 (n = 4) | 21 ± 4 (n = 3) | 54 ± 7 (n = 12) | |
| B maxH (pmol/mg of protein) | 1.6 ± 0.25 (n = 4) | 1.9 ± 0.7 (n = 3) | 0.12 ± 0.02 (n = 12) | |
| % Total | 47% | 41% | ||
| KdL * (pm) | 229 ± 56 (n = 4) | 469 ± 64 (n = 3) | ||
| B maxL (pmol/mg of protein) | 1.8 ± 0.4 (n = 4) | 2.7 ± 0.7 (n = 3) | ||
| % Total | 53% | 59% | ||
The dissociation kinetic parameters k d1 andk d2 were derived from experimental binding data by nonlinear curve fitting as detailed in Materials and Methods. The association rate constant, ka , for [3H]-OFQ was determined from the slope of a plot ofk obs versus [3H]OFQ concentration for association experiments carried out at four concentrations of [3H]OFQ (9). The ka value derived by this method did not differ from the ka value calculated using the pseudo-first-order simplification; thus,ka values for 125I-Tyr14-OFQ binding were determined using pseudo-first-order calculations (9). The equilibrium dissociation constants KdH andKdL were derived from the averaged kinetic values as follows: KdH =k d1/ka andKdL =k d2/ka . The steady-state values for KdH * and KdL *were obtained by nonlinear curve fitting of saturation binding data as detailed in Materials and Methods. All values represent the mean ± standard error of three or four experiments or mean ± range of two experiments, all carried out in duplicate or triplicate.