Table 1.
Maximum GTP hydrolysis and C50 for Arf GAP1 and Arf GAP2
| Maximum GTP Hydrolysis (%) and C50 (nM) | ||||||
|---|---|---|---|---|---|---|
| Vesicles | Lipid content | 0% PIPx | 2.5% PIP2 | 2.5% PI4P | ||
| Extrusion pore | 1.0 μm | 1.0 μm | 0.03 μm | 1.0 μm | 0.03 μm | |
| ArfGAP1 | Max Hydrolysis (%) C50 (nM) |
ND | 98.9±3.0 31.5±2.2 |
98.4±2.3 35.2±14.8 |
ND | ND |
| ArfGAP2 | Max Hydrolysis (%) C50 (nM) |
11 ± 1.6 >500 |
25 ± 2.0*** >500 |
37 ± 5.2***,+ >500 |
6.2 ± 1.2 >500 |
15 ± 1+++ >500 |
| ArfGAP2+coatomer | Max Hydrolysis (%) C50 (nM) |
22 ± 2.0 15 ± 8.6 |
45 ± 2.4^^^ 26 ± 8.4 |
81 ± 3.3^^^,+++ 19 ± 4.8 |
55 ± 3.9^^^ 5.7 ± 1.8@@ |
86 ± 3.2^^^,+++ 7.4 ± 1.2@ |
| ArfGAP2+coatomer+p25 | Max Hydrolysis (%) C50 (nM) |
ND | ND | ND | 47 ± 2.5 1.9 ± 0.7%%% |
84 ± 1.6 0.78 ± 0.2%%% |
The indicated Arf GAPs were titrated into a reaction mixture containing 0.6 μM [α32P]GTP•Arf1 with LUVs containing 10% PI (column labeled 0% PIPx), 7.5% PI and 2.5% PIP2 (column labeled 2.5% PIP2) or 7.5% PI and 2.5% PI4P (column labeled PI4P) and extruded through membranes with pores 0.03 μm or 1.0 μm in diameter, and, as indicated, 124 nM coatomer and 25 μM p25 peptide. The top entry is the maximum percentage of GTP, bound to Arf1, hydrolyzed in a 3 min reaction at 30 °C. The bottom entry is the amount of the indicated Arf GAP needed to achieve 50% of the maximum observed hydrolysis. Arf GAP2 was titrated to a concentration of 3.4 μM in those experiments in which a clear maximum was not achieved. The means ± SD from three experiments are presented. ND, not done. Three ANOVA analysis with Bonferroni post tests were performed. In the first, Arf GAP1 and Arf GAP2 using PIP2 containing vesicles of two different sizes were compared.
indicates less than Arf GAP1 under the same condition, p<0.001. A second ANOVA followed by a Bonferroni post test compared maximum hydrolysis observed with Arf GAP2 and Arf GAP2+ coatomer.
indicates greater than Arf GAP2 with no coatomer, p<0.001
greater than hydrolysis with 1.0 μm, p<0.05
greater than hydrolysis with 1.0 μm vesicle, p<0.001. A third ANOVA analysis compared the C50 values for Arf GAP2 + coatomer with Arf GAP2 + coatomer + p25.
indicates less than C50 with vesicles containing PIP2, p< 0.05;
indicates less than with vesicles containing PIP2, p<0.01;
indicates less than Arf GAP2 + coatomer, p<0.001.