Measurements of ATP in mammalian cells

Manfredi, Giovanni; Yang, Le; Gajewski, Carl D.; Mattiazzi, Marina

doi:10.1016/s1046-2023(02)00037-3

Cited by 204 publications

(148 citation statements)

References 35 publications

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“…36 Briefly, cells were pelleted by centrifugation at 800 g for 3 min at 4 1C, resuspended to 1 Â 10 7 cell ml À1 in buffer A (150 mM KCl, 25 mM Tris-HCl, 2 mM EDTA, 0.1% bovine serum albumin, 10 mM KH 2 PO 4 , 0.1 mM MgCl 2 , pH 7.4). This suspension can be kept on ice for up to 45 min without loss of ATP synthesis activity.…”

Section: Atp Synthesis Ratementioning

confidence: 99%

Selective elimination of mutant mitochondrial genomes as therapeutic strategy for the treatment of NARP and MILS syndromes

et al. 2008

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Mitochondrial diseases are not uncommon, and may result from mutations in both nuclear and mitochondrial DNA (mtDNA). At present, only palliative therapies are available for these disorders, and interest in the development of efficient treatment protocols is high. Here, we demonstrate that in cells heteroplasmic for the T8993G mutation, which is a cause for the NARP and MILS syndromes, infection with an adenovirus, which encodes the mitochondrially targeted R.XmaI restriction endonuclease, leads to selective destruction of mutant mtDNA. This destruction proceeds in a timeand dose-dependent manner and results in cells with significantly increased rates of oxygen consumption and ATP production. The delivery of R.XmaI to mitochondria is accompanied by improvement in the ability to utilize galactose as the sole carbon source, which is a surrogate indicator of the proficiency of oxidative phosphorylation. Concurrently, the rate of lactic acid production by these cells, which is a marker of mitochondrial dysfunction, decreases. We further demonstrate that levels of phosphorylated P53 and gH2ax proteins, markers of nuclear DNA damage, do not change in response to infection with recombinant adenovirus indicating the absence of nuclear DNA damage and the relative safety of the technique. Finally, some advantages and limitations of the proposed approach are discussed.

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Section: Atp Synthesis Ratementioning

confidence: 99%

Selective elimination of mutant mitochondrial genomes as therapeutic strategy for the treatment of NARP and MILS syndromes

et al. 2008

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“…43 N-ATR binding was quantified as nmol C-ATR/mg mitochondrial protein, normalized to citrate synthase (CS) activity. 44 The mitochondrial export of ATP by the ANT carrier too was assayed using a kinetic bioluminescence assay 45 (ATP Bioluminescence Kit CLS II, Roche). Coupled mitochondrial protein (25 mg) were combined with 25 ml of bioluminescent reagent in a 200 ml reaction in a SiriusLuminometer (Berthold Detection Systems).…”

Section: Assays For Ant1 Functionmentioning

confidence: 99%

Adeno-associated virus-mediated gene transfer of the heart/muscle adenine nucleotide translocator (ANT) in mouse

Flierl

Coşkun

et al. 2005

Gene Ther

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“…This mutation is associated with two related syndromes, NARP (neuropathy, ataxia, and retinitis pigmentosa; Holt et al, 1990) and MILS (maternally inherited Leigh syndrome; Santorelli et al, 1993). Importantly, in cytoplasmic hybrid (cybrid) cells containing homoplasmic levels of the mutation, mitochondrial ATP synthesis is reduced by ϳ50 -80% (Manfredi et al, 2002b), but the remainder of the respiratory chain is relatively unaffected. The second mutation is an A3 G transition at position 3243 in the gene encoding tRNA Leu(UUR) .…”

Section: Introductionmentioning

confidence: 99%

“…For the nuclear luciferase (Luc n ), we fused Luc c downstream of the SV-40 large T antigen (accession number NC001669) nuclear localization signal (NLS); this NLS was shown to target recombinant luciferase to the nucleus (Michels et al, 1995). All the chimeric Luc constructs were inserted into the mammalian expression vector pCDNA3.0 (Invitrogen Inc., Carlsbad, CA), as described (Manfredi et al, 2002b). …”

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“…Beetle luciferin (Promega) was added to the cell suspension (final concentration, 2 mM), and light emission was measured in a luminometer (MGM Instruments, Camden, CT) at 5-s intervals until the maximum value of luminescence was reached. To correct for the variability of luciferase expression in each sample, the relative luminescence values in each cell compartment were normalized to the "total potential luminescence" as described above.To assess the total ATP content in cells incubated in the same conditions as above, we used an HPLC-based method (Manfredi et al, 2002b), using appropriate ATP standards. ATP content measured in total cell lysates was expressed as nmoles/mg cellular proteins.…”

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confidence: 99%

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New Insights into the Bioenergetics of Mitochondrial Disorders Using Intracellular ATP Reporters

Gajewski

Yang

Schon

et al. 2003

MBoC

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Mutations in mitochondrial DNA (mtDNA) cause impairment of ATP synthesis. It was hypothesized that high-energy compounds, such as ATP, are compartmentalized within cells and that different cell functions are sustained by different pools of ATP, some deriving from mitochondrial oxidative phosphorylation (OXPHOS) and others from glycolysis. Therefore, an OXPHOS dysfunction may affect different cell compartments to different extents. To address this issue, we have used recombinant forms of the ATP reporter luciferase localized in different cell compartmentsthe cytosol, the subplasma membrane region, the mitochondrial matrix, and the nucleus-of cells containing either wild-type or mutant mtDNA. We found that with glycolytic substrates, both wild-type and mutant cells were able to maintain adequate ATP supplies in all compartments. Conversely, with the OXPHOS substrate pyruvate ATP levels collapsed in all cell compartments of mutant cells. In wild-type cells normal levels of ATP were maintained with pyruvate in the cytosol and in the subplasma membrane region, but, surprisingly, they were reduced in the mitochondria and, to a greater extent, in the nucleus. The severe decrease in nuclear ATP content under "OXPHOS-only" conditions implies that depletion of nuclear ATP plays an important, and hitherto unappreciated, role in patients with mitochondrial dysfunction. INTRODUCTIONThe concept that high-energy compounds are compartmentalized in cells was proposed more than 20 years ago (Erickson-Viitanen et al., 1982a, 1982bSaks et al., 1994). For example, it was suggested that in normal smooth muscle cells the contractile functions are supported by mitochondrial ATP derived from the respiratory chain and oxidative phosphorylation (OXPHOS), whereas the plasma membrane proton pumps are supported by ATP from anaerobic glycolysis (Ishida et al., 1994). In addition, recent studies showed that the import of histones into the nuclei of neonatal cardiomyocytes is strictly dependent on a concerted interaction between mitochondrial ATP synthesis and the trafficking of high-energy phosphoryls (Dzeja et al., 2002).In a technical advance, targeted luciferase has been used as an ATP sensor to investigate the kinetics of the variation of ATP concentration beneath the plasma membrane, in the mitochondria, and in the cytosol of pancreatic ␤-cells in response to glucose stimulation (Kennedy et al., 1999). These experiments demonstrated that in response to the administration of glucose and potassium, ATP levels increased in the plasma membrane of ␤-cells in concert with that in mitochondria, whereas cytosolic ATP showed only a transient increase. On the other hand, studies using the ATP-dependent potassium channel as an ATP sensor showed that in Xenopus oocytes and in cultured mammalian cells there was no gradient between bulk cytosolic ATP and subplasma membrane ATP, suggesting that ATP diffuses freely between these two cell compartments (Gribble et al., 2000).Despite a growing body of evidence that high-energy molecules such as ATP and phos...

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Measurements of ATP in mammalian cells

Cited by 204 publications

References 35 publications

Selective elimination of mutant mitochondrial genomes as therapeutic strategy for the treatment of NARP and MILS syndromes

Selective elimination of mutant mitochondrial genomes as therapeutic strategy for the treatment of NARP and MILS syndromes

Adeno-associated virus-mediated gene transfer of the heart/muscle adenine nucleotide translocator (ANT) in mouse

New Insights into the Bioenergetics of Mitochondrial Disorders Using Intracellular ATP Reporters

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