TABLE 1.
Assay of 6-dehydro-VB-A reductase activity for crude and purified rOrf4 protein
| Protein | Results with the following substrates:
|
Amt of VB-A produced (pmol/min)c | ||
|---|---|---|---|---|
| 6-Dehydro-VB-Aa | NADPHb | NADHb | ||
| Crude (pET-orf4) | + | + | − | 3.51 × 102 |
| Crude (pET-3d) | + | + | − | 0 |
| Purified rOrf4 | + | + | − | 3.34 × 102 |
| Purified rOrf4 | + | − | + | 0 |
| Purified rOrf4 | + | − | − | 0 |
| Purified rOrf4 | − | + | − | 0 |
| No protein | + | + | − | 0 |
a
(±)-6-Dehydro-VB-A was added to a final concentration of 766 μM.
b
NADPH or NADH was added to a final concentration of 5 mM.
c
For assays with crude extracts, 1.31 μg of pET-orf4 protein and 14.6 μg of pET-3d protein were used. For assays with purified rOrf4, 3.0 μg of protein was used. The enzyme activity was measured within the linear range of activity after 0.5 to 20 h of incubation, with a 3-pmol detection limit per assay (31, 32).