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[摘要]：The study of dynamic properties of metabolic and signaling networks is hindered by the lack of methods for imaging metabolic fluxes in individual intact cells. We describe a novel optical approach for measuring the changes of metabolic fluxes in cells, based on a two-substrate competition between a physiological substrate and a fluorogenic reporter substrate. We have constructed a model cell system for a two-step metabolic pathway involved in the metabolism of testosterone. Potent androgen testosterone is converted by steroid 5 alpha-reductase to DHT (5 alpha-dlhydrotestosterone), which is subsequently metabolized to 3 alpha-diol (3 alpha,17 beta-androstanediol) by the reductase AKR1C2 (aldo-ketoreductase 1C2), for which we have previously developed the fluorogenic reporter substrate Coumberone. Despite the medicinal importance of 5 alpha-reductase, there are presently no probes or methods for the continuous activity readout of this enzyme in cells. We show that the activity of 5 alpha-R1 (5 alpha-reductase type 1) can be measured in COS-1 cells via the changes of DHT flux. Our system enables a measurement of 5 alpha-reductase activity in cells, via either fluorimetry or fluorescence microscopy, with a wide dynamic range of activities, and provides a continuous optical assay for evaluation of small molecule inhibitors for this important enzyme. Furthermore, this paper demonstrates a novel optical approach to measuring metabolic flux changes in living cells and expands the utility of fluorogenic enzyme reporter substrates: optical reporters can measure not only the activity of the target enzyme but also the activity of other enzymes upstream in the pathway, for which there are no probes available. |
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