TABLE 1.
ARAP1 PtdIns(3,4,5)P3-dependent Arf GAP activity and PtdIns(3,4,5)P3-ARAP1 association
PtdIns(3,4,5)P3, incorporated into LUVs with a total phospholipid concentration of 500 μm, was titrated into Arf GAP reactions containing 0.03 nm PH1-Ank, 0.03 nm mutant PH1-Ank, or 30 nm ArfGAP-Ank. For enzymatic activity, the reactions contained myrArf5¼GTP as a substrate. The average EC50 (concentration of PtdIns(3,4,5)P3 for half-maximal activity) ± S.E. from at least three independent experiments, estimated from the data in Fig. 5, is presented. The numbers in parentheses represent the number of experiments. For protein association, 1 μm protein was incubated with LUVs with a total phospholipid concentration of 1 mm. The Kd is estimated from the data in Fig. 2. The averages ± S.E. with the number of experiments in parentheses are presented.
| Recombinant protein | PtdIns(3,4,5)P3 dependence of enzymatic activity, EC50 | Lipid-protein association, Kd |
|---|---|---|
| μm | μm | |
| PH1-Ank | 1.6 ± 0.1 (7) | 1.6 ± 0.3 (13) |
| ArfGAP-Ank | >7.5 (3) | >10 (3) |
| [K336N]PH1-Ank | >7.5 (3) | >10 (4) |
| [K347N]PH1-Ank | 4.1 ± 1.7 (3) | 0.5 ± 0.2 (4) |
| [R348Q]PH1-Ank | >7.5 (3) | >10 (4) |
| [R351Q]PH1-Ank | 1.9 ± 0.4 (3) | 0.4 ± 0.1 (3) |
| [R450Q]PH1-Ank | 1.7 ± 0.4 (3) | 2.0 ± 0.4 (3) |
| [K453N]PH1-Ank | 2.7 ± 1.4 (3) | 1.9 ± 0.4 (3) |
| [K455N]PH1-Ank | 1.0 ± 0.2 (3) | 1.1 ± 0.1 (3) |