Table 1.
Initial velocities of Ric-8A mutant guanine nucleotide exchange activity. Assays were conducted as described in Materials and methods section of the Main text. For measurement of v1, reaction buffer (50 M HEPES, pH 8.0, 150 M NaCl, 10 M MgCl2, and 1 M TCEP) at 25°C contained 2 μM Gαi1•GDP, 2 μM Ric-8A and 10 μM GTPγS (initial concentration); for measurement of v2, 2 μM Gαi1•GDP, 2 μM Ric-8A were incubated for 5 min before addition of 10 μM GTPγS.
Ric-8A mutant |
v1* (μM/min) |
v2 (μM/min) |
|---|---|---|
WT |
3.84 (1.45) |
4.66 (1.38) |
P454A |
2.88 (0.80) |
5.39 (0.43) |
V455A |
0.82 (0.05) |
0.25 (0.01) |
T456A |
0.59 (0.04) |
0.24 (0.06) |
G457A |
3.47 (1.11) |
6.02 (1.19) |
R458A |
0.63 (0.07) |
0.37 (0.03 |
V459A |
5.65 (0.68) |
6.85 (1.44) |
E460A |
6.54 (0.83) |
5.36 (1.39) |
E461A |
7.45 (0.65) |
5.06 (0.59) |
K462A |
4.46 (0.92) |
5.15 (0.24) |
P463A |
2.90 (0.69) |
4.78 (1.26) |
P464A |
2.83 (0.94) |
4.73 (1.21) |
N465A |
2.25 (1.03) |
4.56 (0.51) |
P466A |
0.47 (0.01) |
0.34 (0.02) |
M467A |
2.17 (0.06) |
4.57 (0.44) |
E468A |
3.82 (0.55) |
6.23 (0.88) |
G469A |
0.63 (0.08) |
0.26 (0.05) |
(1-452)Ric-8A |
0.86 (0.22) |
0.39 (0.03) |
* v1 for the reaction.
Gαi1•GDP + GTPγS + Ric-8A → Gαi1•GTP + Ric-8A + GDP.
v2 for the reaction.
Gαi1•Ric-8A + GTPγS → Gαi1•GTPγS + Ric-8A.
Values in parentheses are the standard deviation for three independent experiments.