Table 3.
Effect of coatomer on kinetic parameters of Arf GAP2
| A. GTP hydrolysis followed with tryptophan fluorescence | ||||
|---|---|---|---|---|
| 0 coatomer | 5 nM coatomer | 124 nM coatomer | 124 nM coatomer + p25 peptide | |
| Km (μM) | 1.7 ± 0.4 (4) | 0.74 ± 0.22 (3) ** | 0.6 ± 0.2 (6)*** | 0.38 ± 0.1 (3) *** |
| kcat (sec−1) | 0.014 ± 0.001 (4) | 0.043 ± 0.005 (3) | 0.19 ± 0.03 (6)*** | 0.36 ± 0.03 (3)***,+++ |
| B. GTP hydrolysis followed using radioisotopic tracer | ||||
| 0 coatomer | 5 nM coatomer | 124 nM coatomer | 124 nM coatomer + p25 peptide | |
| Km (μM) | >13# (3) | 4.8 ± 0.97 (4) | 1.3 ± 0.39 (3) *** | 1.6 ± 0.24 (3) *** |
| kcat (sec−1) | NC (3) | 0.50 ± 0.043 (4) | 1.9 ± 0.18 (3) *** | 3.5 ± 0.4 (3) ***, +++ |
Initial rate as a function of Arf1•GTP concentration was fit to the Michaelis-Menten equation to estimate the Km and Vmax. The kcat was calculated from the Vmax. The reactions contained the indicated concentration of coatomer and, where indicated, 25 μM p25 peptide. A. GAP assayed by changes in fluorescence. Arf1•GTP to Arf1•GDP conversion was followed using tryptophan fluorescence. The parameters are the means and SD from fitting data presented in Figures 5 and 8. The number of experiments is indicated in parentheses. ** indicates different than 0 coatomer, p<0.01; *** indicates different than 0 coatomer, p< 0.001; +++, indicates different than 124 nM coatomer alone, p<0.001. B. GAP reaction followed by conversion of [α32P]GTP to [α32P]GDP. Data from three experiments presented in Figures 5 and 8 were fit to the Michaelis-Menten equation. The parameter estimates are presented as means ± SD for the number of experiments indicated in parentheses. #, did not saturate with reaction velocity linearly related to Arf1•GTP concentration in the range of 0.5 to 13 μM. NC, Not calculated because did not achieve saturation. ***, different than 5 nM coatomer, p<0.001, +++, indicates different than 124 nM coatomer alone, p<0.001.