TABLE 2.
Activation of RetGC1 by selected GCAP1 mutants
This material was compiled from titrations exemplified in Figs. 2 and 3 and Ref. 56. The K1/2 GCAP and amax (means ± S.E.) values are from the fit of the data sum from n independent trials, assuming the Michaelis function a = amax [GCAP]/(K1/2 GCAP + [GCAP]), where a is the activity of RetGC in the assay, amax is the maximal activity of RetGC, [GCAP] is the concentration of GCAP1, and K1/2 GCAP is the GCAP concentration required for half-maximal activation. The data were fitted using KaleidaGraph 4.0 utilizing a conventional Levenberg-Marquardt algorithm of nonlinear least-squares routines. ND, not determined (the activity was too low to reliably extract the parameters).
| GCAP1 | K1/2 GCAP | amax |
|---|---|---|
| WT | 1.6 ± 0.1 | 27 ± 1.6 (n = 5) |
| E17K | 8.3 ± 0.1 | 18 ± 0.3 (n = 2) |
| C18T,C29T,C106T,C125T | 12 ± 2 | 29 ± 1 (n = 3) |
| C18T,C106T,C125T | 1.2 ± 0.2 | 25 ± 0.2 (n = 3) |
| H19R | 4.5 ± 0.4 | 18 ± 1 (n = 2) |
| K23D | 25 ± 4a | 9 ± 0.9 (n = 2) |
| M26R | ND | ND (n = 2) |
| T27K | 3.3 ± 0.5 | 28 ± 2 (n = 2) |
| T27E | 2.9 ± 0.1 | 28 ± 0.4 (n = 2) |
| E28R | 29 ± 4a | 28 ± 3 (n = 2) |
| P30Y | ND | ND (n = 5) |
| S31Y | 60 ± 19a | 10 ± 3 (n = 2) |
| G32N | 13 ± 3a | 39 ± 12 (n = 2) |
| K46D | 9.7 ± 0.9 | 31 ± 1.4 (n = 2) |
| F73E | ND | ND (n = 2) |
| M74K | 7.1 ± 0.5 | 17 ± 1 (n = 2) |
| V77E | ND | ND (n = 3) |
| A78E | ND | ND (n = 3) |
| K85E | ND | ND (n = 2) |
| K87D | 15 ± 1 | 19 ± 0.4 (n = 2) |
| W94A | ND | ND (n = 2) |
| K97S | 13 ± 1 | 12 ± 1 (n = 2) |
| S141Y | 1.6 ± 0.1 | 27 ± 0.4 (n = 2) |
| S152E | 7.2 ± 0.6 | 20 ± 0.6 (n = 2) |
| L153R | 3.2 ± 0.1 | 27 ± 0.5 (n = 2) |
| L166R | 3.9 ± 0.2 | 28 ± 0.7 (n = 2) |
a Because of the dramatic loss of affinity for the target and limitations of the fit method, the error was larger for those mutants in which K1/2 GCAP was similar to or above the maximal GCAP concentration in the assay, and the measured a was much lower than amax.