A rapid HPLC method for determination of adenylate energy charge

Viarengo, Aldo; Secondini, A.; Scoppa, P.; Orunesu, M

doi:10.1007/bf01946400

Cited by 13 publications

(5 citation statements)

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“…In addition, this mode of ATP usage may allow TRiC to function under low-energy conditions. Under steady-state conditions, the ATP concentration in yeast and mammalian cells has been estimated to be in the low millimolar range (Hara and Mori, 2006; Richard et al, 1996; Viarengo et al, 1986). However, the ATP concentration has been shown to oscillate in vivo in both mammalian cells (Bertram et al, 2007) and Saccharomyces cerevisiae (Richard, 2003) depending on the physiological state.…”

Section: Discussionmentioning

confidence: 99%

A Gradient of ATP Affinities Generates an Asymmetric Power Stroke Driving the Chaperonin TRIC/CCT Folding Cycle

et al. 2012

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SUMMARY The eukaryotic chaperonin TRiC/CCT uses ATP cycling to fold many essential proteins that other chaperones cannot fold. This 1 MDa hetero-oligomer consists of two identical stacked rings assembled from eight paralogous subunits, each containing a conserved ATP-binding domain. Here, we report a dramatic asymmetry in the ATP utilization cycle of this ring-shaped chaperonin, despite its apparently symmetric architecture. Only four of the eight different subunits bind ATP at physiological concentrations. ATP binding and hydrolysis by the low-affinity subunits is fully dispensable for TRiC function in vivo. The conserved nucleotide-binding hierarchy among TRiC subunits is evolutionarily modulated through differential nucleoside contacts. Strikingly, high-and low-affinity subunits are spatially segregated within two contiguous hemispheres in the ring, generating an asymmetric power stroke that drives the folding cycle. This unusual mode of ATP utilization likely serves to orchestrate a directional mechanism underlying TRiC/CCT’s unique ability to fold complex eukaryotic proteins.

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Section: Discussionmentioning

confidence: 99%

A Gradient of ATP Affinities Generates an Asymmetric Power Stroke Driving the Chaperonin TRIC/CCT Folding Cycle

et al. 2012

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“…Most current measurements of ATP in cells use extraction of the cell content and measure the concentration of ATP in the extract by various off-line methods such as HPLC (9), luciferase (10), or other enzyme-based methods (11). A few protein-based sensors exist (6,7,(12)(13)(14), which in principle allow for time-resolved measurements of intracellular ATP or ADP, but some of these methods entail expression of the sensor molecule in situ, which is not always possible.…”

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confidence: 99%

Time-resolved Measurements of Intracellular ATP in the Yeast Saccharomyces cerevisiae using a New Type of Nanobiosensor

Özalp

Pedersen

Nielsen

et al. 2010

Journal of Biological Chemistry

106

109

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Adenosine 5-triphosphate is a universal molecule in all living cells, where it functions in bioenergetics and cell signaling. To understand how the concentration of ATP is regulated by cell metabolism and in turn how it regulates the activities of enzymes in the cell it would be beneficial if we could measure ATP concentration in the intact cell in real time. Using a novel aptamer-based ATP nanosensor, which can readily monitor intracellular ATP in eukaryotic cells with a time resolution of seconds, we have performed the first on-line measurements of the intracellular concentration of ATP in the yeast Saccharomyces cerevisiae. These ATP measurements show that the ATP concentration in the yeast cell is not stationary. In addition to an oscillating ATP concentration, we also observe that the concentration is high in the starved cells and starts to decrease when glycolysis is induced. The decrease in ATP concentration is shown to be caused by the activity of membrane-bound ATPases such as the mitochondrial F 0 F 1 ATPase-hydrolyzing ATP and the plasma membrane ATPase (PMA1). The activity of these two ATPases are under strict control by the glucose concentration in the cell. Finally, the measurements of intracellular ATP suggest that 2-deoxyglucose (2-DG) may have more complex function than just a catabolic block. Surprisingly, addition of 2-DG induces only a moderate decline in ATP. Furthermore, our results suggest that 2-DG may inhibit the activation of PMA1 after addition of glucose.Adenosine 5Ј-triphosphate (ATP) is a highly important biomolecule in living cells: It plays a central role in cell energy metabolism and also serves directly or indirectly in a number of cell signaling processes (1-3). The intracellular concentration of ATP is believed to oscillate in some eukaryotic cells, e.g. in ␤-cells (4) and in cells of the yeast Saccharomyces cerevisiae (5). However, changes in cytoplasmic ATP concentration with high time resolution have so far only been measured in a few circumstances (6, 7), mainly because methods for such continuous measurements are not generally available, or, in the case of NMR, require very high densities of cells or tissue (8).The lack of time-resolved measurements has prohibited the understanding of how the level of ATP and other intracellular metabolites are regulated in the cell and how ATP in turn regulates a number of cellular processes. Most current measurements of ATP in cells use extraction of the cell content and measure the concentration of ATP in the extract by various off-line methods such as HPLC (9), luciferase (10), or other enzyme-based methods (11). A few protein-based sensors exist (6,7,(12)(13)(14), which in principle allow for time-resolved measurements of intracellular ATP or ADP, but some of these methods entail expression of the sensor molecule in situ, which is not always possible. Hence, although it is expected that the intracellular concentration of ATP is anything but stationary, this has not been verified by real-time measurements, except in a few cases where ...

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“…Analysis of particulate adenylates for determining biomass is also feasible since common extractants such as Tris and bicarbonate buffers (e.g., see reference 14) do not interfere with the adenylate chromatography. Given the low adenylate detection limits possible by monitoring A254 to A260, derivitization and fluorescence detection (e.g., see reference 33) Downloaded from may be unnecessary (see also Viarengo et al [32] for a gradient-based absorbance method). Finally, other solutes of ecological interest, including glucose, galactose, gluconate, pyruvate, and succinate, are amenable to analysis by using simple modifications (e.g., enzyme substitution, ADP analysis) of the approach described here.…”

Section: Resultsmentioning

confidence: 99%

Measurement of Acetate Concentrations in Marine Pore Waters by Using an Enzymatic Approach

King

1991

Appl Environ Microbiol

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Acetate concentrations in marine and freshwater matrices were measured by an enzymatic technique which coupled the synthesis of acetyl coenzyme A to AMP production. The resulting AMP was assayed by a sensitive and relatively rapid high-pressure liquid chromatography method, using an aqueous, isocratic mobile phase for elution. The method was insensitive to the presence of seawater salts and required no sample prepurification or distillation. Propionate caused a minor, but statistically insignificant, interference when equimolar with acetate; butyrate caused no interference, even at relatively high concentrations. Detection limits for acetate were approximately 100 nM with a precision of about 5%. Pore waters from two intertidal sediments contained approximately 1 to 12 μM acetate; the concentrations were linearly but inversely correlated with porewater sulfate.

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A rapid HPLC method for determination of adenylate energy charge

Abstract: A simple procedure is described for separation and analysis of adenine nucleotides in tissue extracts, utilizing anion exchange HPLC. Determination of AMP, ADP, and ATP takes 10 min per sample.

Cited by 13 publications

References 5 publications

A Gradient of ATP Affinities Generates an Asymmetric Power Stroke Driving the Chaperonin TRIC/CCT Folding Cycle

A Gradient of ATP Affinities Generates an Asymmetric Power Stroke Driving the Chaperonin TRIC/CCT Folding Cycle

Time-resolved Measurements of Intracellular ATP in the Yeast Saccharomyces cerevisiae using a New Type of Nanobiosensor

Measurement of Acetate Concentrations in Marine Pore Waters by Using an Enzymatic Approach

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