TABLE 2.
The effect of inositol polyphosphates on AtMRP5-mediated inositol hexakisphosphate transport
The putative inhibitors and/or competitors were added to the reaction mix consisting of transport buffer, 1 mm dithiothreitol, 5 mm ATP, 10 mm MgCl2, 10 mm creatine-phosphate, and 100 μg/ml creatine kinase at the concentration indicated above. Yeast microsomes were added to start the transport assay. The assays were performed at room temperature. After 5.5 min, inositol hexakisphosphate uptake was terminated by the transfer of three aliquots/repetition onto 0.45-μm-diameter pore size nitrocellulose filters. The putative inhibitors and/or competitors were always tested using the same vesicle preparation. ATP-dependent AtMRP5-mediated transport was considered as 100%. The reaction mix contained labeled inositol hexakisphosphate at 1.85 kBq/nitrocellulose filter.
| Condition | Percentage | Repetitions |
|---|---|---|
| + ATP | 100 | 6 |
| + 600 nm | ||
| Ins(1,4,5)P3 | 111.7 ± 17.6 | 3 |
| Ins(1,3,4,6)P4 | 104.1 ± 18.4 | 3 |
| Ins(1,4,5,6)P4 | 100.6 ± 16.8 | 3 |
| Ins(1,3,4,5,6)P5 | 80.5 ± 6.6 | 2 |
| Scyllo InsP6 | 119.8 ± 12.5 | 3 |
| Ins(1,2,3,4,5,6)P6 | 52.7 ± 1.8 | 2 |
| Estradiol glucuronide | 122.6 ± 16.2 | 3 |
| +1200 nm | ||
| Ins(3,4,6)P3 | 157.3 ± 3.8 | 3 |
| Ins(1,3,4,6)P4 | 100.8 ± 30.3 | 2 |
| Ins(1,4,5,6)P4 | 80.2 ± 20.2 | 3 |
| Ins(1,3,4,5,6)P5 | 61.0 ± 7.1 | 2 |
| Scyllo InsP6 | 57.7 ± 13.6 | 2 |
| Ins(1,2,3,4,5,6)P6 | 0 ± 0 | 2 |
| Estradiol glucuronide | 92.4 ± 4.9 | 3 |