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Determination of the Cytosolic NADPH/NADP Ratio in Saccharomyces cerevisiae using Shikimate Dehydrogenase as Sensor Reaction.

Zhang J, ten Pierick A, van Rossum HM, Seifar RM, Ras C, Daran JM, Heijnen JJ, Wahl SA - Sci Rep (2015)

Bottom Line: In response to a glucose pulse, the cytosolic NADPH/NADP ratio first increased very rapidly and restored the steady state ratio after 3 minutes.In contrast to this dynamic observation, the whole cell NADPH/NADP ratio remained nearly constant.The novel cytosol NADPH/NADP measurements provide new insights into the thermodynamic driving forces for NADP(H)-dependent reactions, like amino acid synthesis, product pathways like fatty acid production or the mevalonate pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Delft University of Technology, Delft, 2628BC, The Netherlands.

ABSTRACT
Eukaryotic metabolism is organised in complex networks of enzyme catalysed reactions which are distributed over different organelles. To quantify the compartmentalised reactions, quantitative measurements of relevant physiological variables in different compartments are needed, especially of cofactors. NADP(H) are critical components in cellular redox metabolism. Currently, available metabolite measurement methods allow whole cell measurements. Here a metabolite sensor based on a fast equilibrium reaction is introduced to monitor the cytosolic NADPH/NADP ratio in Saccharomyces cerevisiae: NADP + shikimate ⇄ NADPH + H(+) + dehydroshikimate. The cytosolic NADPH/NADP ratio was determined by measuring the shikimate and dehydroshikimate concentrations (by GC-MS/MS). The cytosolic NADPH/NADP ratio was determined under batch and chemostat (aerobic, glucose-limited, D = 0.1 h(-1)) conditions, to be 22.0 ± 2.6 and 15.6 ± 0.6, respectively. These ratios were much higher than the whole cell NADPH/NADP ratio (1.05 ± 0.08). In response to a glucose pulse, the cytosolic NADPH/NADP ratio first increased very rapidly and restored the steady state ratio after 3 minutes. In contrast to this dynamic observation, the whole cell NADPH/NADP ratio remained nearly constant. The novel cytosol NADPH/NADP measurements provide new insights into the thermodynamic driving forces for NADP(H)-dependent reactions, like amino acid synthesis, product pathways like fatty acid production or the mevalonate pathway.

No MeSH data available.


Intracellular SA, DHS, NADPH, NADP amounts, cytosolic and whole cell NADPH/NADP ratio during the pulse experiments.Data points before t = 0 represent the state 5 mins after the SA addition, just before the glucose perturbation.
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f4: Intracellular SA, DHS, NADPH, NADP amounts, cytosolic and whole cell NADPH/NADP ratio during the pulse experiments.Data points before t = 0 represent the state 5 mins after the SA addition, just before the glucose perturbation.

Mentions: The extracellular responses of glucose, SA and DHS are shown in Fig. 3. The average uptake rate of glucose in the first 10 minutes was 0.54 μmol/(gDW∙s), and comparable to previous pulse experiments40, which is about 1.6 times higher than the steady state (0.33 μmol/(gDW∙s)). There was no considerable changes of extracellular SA in response to the glucose pulse, and the extracellular DHS decreased from 1.72 to 1.10 during the first seconds of the glucose pulse, and increased back to pre-pulse value in two minutes. The intracellular concentration of SA changed about 2 fold (Fig. 4, from 0.22 μmol/gDW to 0.41 μmol/gDW). This shows that the SA transport is not affected by changing intracellular SA level, confirming that SA transport is the irreversible symport mechanism.


Determination of the Cytosolic NADPH/NADP Ratio in Saccharomyces cerevisiae using Shikimate Dehydrogenase as Sensor Reaction.

Zhang J, ten Pierick A, van Rossum HM, Seifar RM, Ras C, Daran JM, Heijnen JJ, Wahl SA - Sci Rep (2015)

Intracellular SA, DHS, NADPH, NADP amounts, cytosolic and whole cell NADPH/NADP ratio during the pulse experiments.Data points before t = 0 represent the state 5 mins after the SA addition, just before the glucose perturbation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4525286&req=5

f4: Intracellular SA, DHS, NADPH, NADP amounts, cytosolic and whole cell NADPH/NADP ratio during the pulse experiments.Data points before t = 0 represent the state 5 mins after the SA addition, just before the glucose perturbation.
Mentions: The extracellular responses of glucose, SA and DHS are shown in Fig. 3. The average uptake rate of glucose in the first 10 minutes was 0.54 μmol/(gDW∙s), and comparable to previous pulse experiments40, which is about 1.6 times higher than the steady state (0.33 μmol/(gDW∙s)). There was no considerable changes of extracellular SA in response to the glucose pulse, and the extracellular DHS decreased from 1.72 to 1.10 during the first seconds of the glucose pulse, and increased back to pre-pulse value in two minutes. The intracellular concentration of SA changed about 2 fold (Fig. 4, from 0.22 μmol/gDW to 0.41 μmol/gDW). This shows that the SA transport is not affected by changing intracellular SA level, confirming that SA transport is the irreversible symport mechanism.

Bottom Line: In response to a glucose pulse, the cytosolic NADPH/NADP ratio first increased very rapidly and restored the steady state ratio after 3 minutes.In contrast to this dynamic observation, the whole cell NADPH/NADP ratio remained nearly constant.The novel cytosol NADPH/NADP measurements provide new insights into the thermodynamic driving forces for NADP(H)-dependent reactions, like amino acid synthesis, product pathways like fatty acid production or the mevalonate pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Delft University of Technology, Delft, 2628BC, The Netherlands.

ABSTRACT
Eukaryotic metabolism is organised in complex networks of enzyme catalysed reactions which are distributed over different organelles. To quantify the compartmentalised reactions, quantitative measurements of relevant physiological variables in different compartments are needed, especially of cofactors. NADP(H) are critical components in cellular redox metabolism. Currently, available metabolite measurement methods allow whole cell measurements. Here a metabolite sensor based on a fast equilibrium reaction is introduced to monitor the cytosolic NADPH/NADP ratio in Saccharomyces cerevisiae: NADP + shikimate ⇄ NADPH + H(+) + dehydroshikimate. The cytosolic NADPH/NADP ratio was determined by measuring the shikimate and dehydroshikimate concentrations (by GC-MS/MS). The cytosolic NADPH/NADP ratio was determined under batch and chemostat (aerobic, glucose-limited, D = 0.1 h(-1)) conditions, to be 22.0 ± 2.6 and 15.6 ± 0.6, respectively. These ratios were much higher than the whole cell NADPH/NADP ratio (1.05 ± 0.08). In response to a glucose pulse, the cytosolic NADPH/NADP ratio first increased very rapidly and restored the steady state ratio after 3 minutes. In contrast to this dynamic observation, the whole cell NADPH/NADP ratio remained nearly constant. The novel cytosol NADPH/NADP measurements provide new insights into the thermodynamic driving forces for NADP(H)-dependent reactions, like amino acid synthesis, product pathways like fatty acid production or the mevalonate pathway.

No MeSH data available.