查看文章

The coenzyme NAD plays a major role in metabolism as a key redox carrier and signaling molecule but current measurement techniques cannot distinguish between different compartment pools, between free and protein‐bound forms and/or between NAD(H) and NADP(H). Local free NAD/NADH ratios can be determined from product/substrate ratios of suitable near‐equilibrium redox reactions but the application of this principle is often precluded by uncertainties regarding enzyme activity, localization and coenzyme specificity of dehydrogenases. In Saccharomyces cerevisiae, we circumvented these issues by expressing a bacterial mannitol‐1‐phosphate 5‐dehydrogenase and determining the cytosolic free NAD/NADH ratio from the measured [fructose‐6‐phosphate]/[mannitol‐1‐phosphate] ratio. Under aerobic glucose‐limited conditions we estimated a cytosolic free NAD/NADH ratio between 101(±14) and …