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Semisynthetic biosensors for mapping cellular concentrations of nicotinamide adenine dinucleotides.

ABSTRACT: We introduce a new class of semisynthetic fluorescent biosensors for the quantification of free nicotinamide adenine dinucleotide (NAD+) and ratios of reduced to oxidized nicotinamide adenine dinucleotide phosphate (NADPH/NADP+) in live cells. Sensing is based on controlling the spatial proximity of two synthetic fluorophores by binding of NAD(P) to the protein component of the sensor. The sensors possess a large dynamic range, can be excited at long wavelengths, are pH-insensitive, have tunable response range and can be localized in different organelles. Ratios of free NADPH/NADP+ are found to be higher in mitochondria compared to those found in the nucleus and the cytosol. By recording free NADPH/NADP+ ratios in response to changes in environmental conditions, we observe how cells can react to such changes by adapting metabolic fluxes. Finally, we demonstrate how a comparison of the effect of drugs on cellular NAD(P) levels can be used to probe mechanisms of action.

SUBMITTER: Sallin O

PROVIDER: S-EPMC5990361 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Semisynthetic biosensors for mapping cellular concentrations of nicotinamide adenine dinucleotides.

Sallin Olivier O Reymond Luc L Gondrand Corentin C Raith Fabio F Koch Birgit B Johnsson Kai K

eLife 20180529

We introduce a new class of semisynthetic fluorescent biosensors for the quantification of free nicotinamide adenine dinucleotide (NAD<sup>+</sup>) and ratios of reduced to oxidized nicotinamide adenine dinucleotide phosphate (NADPH/NADP<sup>+</sup>) in live cells. Sensing is based on controlling the spatial proximity of two synthetic fluorophores by binding of NAD(P) to the protein component of the sensor. The sensors possess a large dynamic range, can be excited at long wavelengths, are pH-ins ...[more]

PMID: 29809136

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Project description:1. The redox state of the NAD couple of rat liver mitochondria, as measured by the [beta-hydroxybutyrate]/[acetoacetate] ratio, rapidly changed in the direction of oxidation during the preparation of homogenates in a saline medium. The value of the [beta-hydroxybutyrate]/[acetoacetate] ratio fell from 2.3 to 0.15 in 10min. EDTA diminished the fall and succinate prevented it. 2. The redox state of the rat liver cytoplasm, as measured by the [lactate]/[pyruvate] ratio, changed slightly in the direction of reduction during the preparation of homogenate. This was prevented by succinate. 3. In unsupplemented homogenates the differences in the redox states of mitochondria and cytoplasm decreased. Succinate and EDTA together maintained the differences within the physiological range. A measure of the ability of the mitochondria to maintain different redox states in mitochondria and cytoplasm is the value of the expression [lactate][acetoacetate]/[pyruvate][beta-hydroxybutyrate]. If there are no differences in the redox states of the NAD in the two cell compartments the value of the expression is 444 at 37 degrees . The value in the intact rat liver is between 4.7 and 21. 4. alpha-Oxoglutarate or glutamate were still more effective than succinate in maintaining high [beta-hydroxybutyrate]/[acetoacetate] ratios in the homogenates because these substrates supply a reducing agent of NAD(+) and, through succinate, an inhibitor of the oxidation of NADH. 5. When supplemented with alpha-oxoglutarate and EDTA, homogenates readily adjust the redox state of the beta-hydroxybutyrate dehydrogenase system after it has been upset by the addition of either acetoacetate or beta-hydroxybutyrate. 6. Amytal and rotenone raised the value of the [beta-hydroxybutyrate]/[acetoacetate] ratio. This is taken to indicate that the reduction of acetoacetate in the homogenates was not an energy-linked process. 7. 2,4-Dinitrophenol shifted the [beta-hydroxybutyrate]/[acetoacetate] ratio in the presence of succinate in favour of oxidation because it inhibited the oxidation of succinate and accelerated the oxidation of NADH. 8. Rotenone increased the rate of ketone-body formation of liver homogenates, though it decreased the rate of oxygen uptake.

Dataset Information

Semisynthetic biosensors for mapping cellular concentrations of nicotinamide adenine dinucleotides.

Publications

Semisynthetic biosensors for mapping cellular concentrations of nicotinamide adenine dinucleotides.

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