Table 2.
Reconstitution assays of Mg-chelatase activity in soluble chloroplast extracts from barley Chlorina mutants
| Plastid stroma | Wild-type subunits supplied | Mg-chelatase
activity
|
|
|---|---|---|---|
| pmol/30 min | pmol/min/mg protein | ||
| Wild-type | None | 164 ± 64 | 17.6 ± 3.0 |
| F, H | 318 ± 11 | 19.9 ± 2.7 | |
| F, G | 148 ± 66 | 4.78 ± 3.43 | |
| None | F, H | 18.6 | 2.38 |
| F, G | 0.0 | 0.0 | |
| F, G, H | 307 | 10.9 | |
| Chlorina-125 (Clo/Clo) | None | 0.0 | 0.0 |
| F, H | 49.7 | 4.04 | |
| F, G | 0.0 | 0.0 | |
| Chlorina-157 (Clo/Clo) | None | 0.0 | 0.0 |
| F, H | 64.4 | 6.71 | |
| F, G | 0.0 | 0.0 | |
| Chlorina-161 (Clo/Clo) | None | 0.0 | 0.0 |
| F, H | 141 | 9.15 | |
| F, G | 0.0 | 0.0 | |
| Chlorina-125 (Clo/clo) | None | 40.6 | 4.51 |
| F, H | 317 | 18.9 | |
| F, G | 35.5 | 1.21 | |
| Chlorina-157 (Clo/clo) | None | 201 | 16.7 |
| F, H | 294 | 14.9 | |
| F, G | 152 | 4.70 | |
| Chlorina-161 (Clo/clo) | None | 81.2 | 6.59 |
| F, H | 314 | 15.6 | |
| F, G | 71.0 | 2.16 | |
Wild-type 42-kDa subunits (H) were separated into a high-speed supernatant from pelleted wild-type 70-kDa subunits (G). The 140-kDa subunit (F) was present in both fractions. These wild-type subunits were supplied to reconstitution assays of Mg-chelatase activity. The amounts of protein used in 50-μl assays were 260 μg of supernatant (F, H), 680 μg of pellet (F, G), 150 μg of Chlorina-125 (Clo/Clo), 300 μg of Chlorina-125 (Clo/clo), 159 μg of Chlorina-125 (clo/clo), 60 μg of Chlorina-157 (Clo/Clo), 399 μg of Chlorina-157 (Clo/clo), 420 μg of Chlorina-157 (clo/clo), 255 μg of Chlorina-161 (Clo/Clo), 411 μg of Chlorina-161 (Clo/clo), and 300 μg of Chlorina-161 (clo/clo).