TABLE 3.
Effect of the D777N suppressor mutations on the kinetic parameters of LTC4 uptake in vacuolar membrane vesicles
| Mutant | Km (ATP)a | Vmaxa |
|---|---|---|
| Wild type | 0.05 ± 0.01 | 4.40 ± 0.31 |
| D777N | 1.42 ± 0.18 | 1.69 ± 0.24 |
| D777N/V543I | 1.09 ± 0.28 | 1.15 ± 0.04 |
| D777N/F565L | 1.44 ± 0.30 | 0.58 ± 0.04 |
| D777N/S674L | 1.37 ± 0.26 | 1.21 ± 0.05 |
| D777N/A1003V | 1.55 ± 0.46 | 1.64 ± 0.13 |
| D777N/A1021T | 1.14 ± 0.20 | 1.28 ± 0.12 |
| D777N/A1021V | 1.71 ± 0.39 | 1.47 ± 0.15 |
| D777N/N1027D | 1.16 ± 0.18 | 2.49 ± 0.34 |
| D777N/Q1107R | 1.07 ± 0.09 | 1.48 ± 0.13 |
| D777N/G1207D | 1.06 ± 0.36 | 0.8 ± 0.06 |
| D777N/G1207S | 1.12 ± 0.27 | 2.33 ± 0.14 |
| D777N/S1212L | 1.39 ± 0.12 | 1.36 ± 0.13 |
| D777N/W1225C | —b | — |
| D777N/R1415G | 1.65 ± 0.44 | 2.02 ± 0.27 |
a
To determine the apparent Km for ATP (mM), the initial rate of LTC4 uptake in vacuolar membrane vesicles was assayed with 50 nM LTC4 and ATP concentrations ranging from 0.035 to 6 mM (see Materials and Methods). The Vmax (nmol · min−1 · mg−1) was determined with 10 mM ATP and LTC4 concentrations ranging from 0.1 to 3 μM. Data were fitted to the Michaelis-Menten equation using Prism 2.0 GraphPad Software. Values are the average ± standard deviation of duplicate determinations in two independently isolated vacuolar membrane preparations.
b
No detectable transport activity for LTC4.