Table 1.
Probe 1 enabled the quantitative evaluation of a physiological process, DHT reduction to 3α-diol
| Substrate |
KM |
KM,app |
|
|---|---|---|---|
| Purified protein, fluorimetric | Intact cells, fluorimetric | Intact cells, radiometric | |
| Reporter probe 1 | 3.0 ± 0.2 | 3.0 ± 0.4 | — |
| DHT | 1.3 ± 0.2* | 1.3 ± 0.2 | 1.3 ± 0.4 |
All values are given as micromolar concentrations. Through a two-substrate competitive assay between physiological substrate DHT and reporter substrate 1, the KM of DHT was measured directly in living cells. The conventional assay for cellular kinetics that requires discontinuous analysis of radiolabeled metabolites is in excellent agreement with the fluorimetric measurement.
*In agreement with radiometric assay (1.4 ± 0.2 μM).