A sensitive fluorimetric assay for pyruvate

Zhu, Aiping; Romero, Roberto; Petty, Howard R.

doi:10.1016/j.ab.2009.09.017

Cited by 63 publications

(54 citation statements)

References 22 publications

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“…3g) 37 . At concentrations of pyruvate greater than 0.3 mM, acidic pH impaired the ability of LDH to reduce pyruvate due to enhanced substrate inhibition as previously described (Fig.…”

Section: Resultsmentioning

confidence: 99%

L-2-Hydroxyglutarate production arises from noncanonical enzyme function at acidic pH

et al. 2017

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The metabolite 2-hydroxyglutarate (2HG) can be produced as either a D(R)- or L(S)- enantiomer, each of which inhibits alpha-ketoglutarate (αKG)-dependent enzymes involved in diverse biologic processes. Oncogenic mutations in isocitrate dehydrogenase produce D-2HG, which causes a pathologic blockade in cell differentiation. On the other hand, oxygen limitation leads to accumulation of L-2HG, which can facilitate physiologic adaptation to hypoxic stress in both normal and malignant cells. Here we demonstrate that purified lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) catalyze stereospecific production of L-2HG via ‘promiscuous’ reduction of the alternative substrate αKG. Acidic pH enhances production of L-2HG by promoting a protonated form of αKG that binds to a key residue in the substrate-binding pocket of LDHA. Acid-enhanced production of L-2HG leads to stabilization of hypoxia-inducible factor 1 alpha (HIF-1α) in normoxia. These findings offer insights into mechanisms whereby microenvironmental factors influence production of metabolites that alter cell fate and function.

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“…3g) 37 . At concentrations of pyruvate greater than 0.3 mM, acidic pH impaired the ability of LDH to reduce pyruvate due to enhanced substrate inhibition as previously described (Fig.…”

Section: Resultsmentioning

confidence: 99%

L-2-Hydroxyglutarate production arises from noncanonical enzyme function at acidic pH

et al. 2017

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“…Pyruvate was extracted as described by Zhu et al (35). Briefly, 5 ϫ 10 8 parasites were incubated in HBS buffer or in glucose-free HBS buffer plus 5 mM 2-deoxy-Dglucose for 1 h at 28°C.…”

Section: Chemicalmentioning

confidence: 99%

“…The supernatant was collected after centrifugation at 10,000 ϫ g for 5 min. Pyruvate was measured using a fluorimetric assay (35) based on the oxidation of pyruvate by pyruvate oxidase. The hydrogen peroxide generated reacts with nonfluorescent Amplex Red at a 1:1 stoichiometry to form the red fluorescent product resorufin.…”

Section: Chemicalmentioning

confidence: 99%

Increased Glycolytic ATP Synthesis Is Associated with Tafenoquine Resistance inLeishmania major

Manzano

Carvalho

Pérez‐Victoria

et al. 2011

Antimicrob Agents Chemother

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Tafenoquine (TFQ), an 8-aminoquinoline used to treat and prevent Plasmodium infections, could represent an alternative therapy for leishmaniasis. Indeed, TFQ has shown significant leishmanicidal activity both in vitro and in vivo, where it targets Leishmania mitochondria and activates a final apoptosis-like process. In order not to jeopardize the life span of this potential antileishmania drug, it is important to determine the likelihood that Leishmania will develop resistance to TFQ and the mechanisms of resistance induced. To address this issue, a TFQ-resistant Leishmania major promastigote line (R4) was selected. This resistance, which is unstable in a drug-free medium (revertant line), was maintained in intramacrophage amastigote forms, and R4 promastigotes were found to be cross-resistant to other 8-aminoquinolines. A decreased TFQ uptake, which is probably associated with an alkalinization of the intracellular pH rather than drug efflux, was observed for both the R4 and revertant lines. TFQ induces a decrease in ATP synthesis in all Leishmania lines, although total ATP levels were maintained at higher values in R4 parasites. In contrast, ATP synthesis by glycolysis was significantly increased in R4 parasites, whereas mitochondrial ATP synthesis was similar to that in wild-type parasites. We therefore conclude that increased glycolytic ATP synthesis is the main mechanism underlying TFQ resistance in Leishmania.

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“…Aiming at the optimization of the assay in the fluorescence mode, two critical parameters were further investigated: the concentration of AR in the reaction mixture and the incubation time before recording fluorescence signals. The nonfluorescent AR reacts with H 2 O 2 in a stoichiometric 1:1 ratio, catalyzed by HRP, to form the highly fluorescent product resorufin [19,20]. However, as has been shown by others [20], at excess levels of H 2 O 2 resorufin can be further oxidized to the nonfluorescent compound resazurin.…”

mentioning

confidence: 88%

“…The nonfluorescent AR reacts with H 2 O 2 in a stoichiometric 1:1 ratio, catalyzed by HRP, to form the highly fluorescent product resorufin [19,20]. However, as has been shown by others [20], at excess levels of H 2 O 2 resorufin can be further oxidized to the nonfluorescent compound resazurin. Therefore, the concentration of AR should be kept at least five times higher than that of H 2 O 2 .…”

mentioning

confidence: 88%

An Amplex Red-based fluorometric and spectrophotometric assay for l-asparaginase using its natural substrate

Karamitros

Lim

Konrad

2014

Analytical Biochemistry

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Leukemia L-Aspartate oxidase Coupled enzyme assay Amplex Red a b s t r a c tWe report on the development of a sensitive real-time assay for monitoring the activity of L-asparaginase that hydrolyzes L-asparagine to L-aspartate and ammonia. In this method, L-aspartate is oxidized by Laspartate oxidase to iminoaspartate and hydrogen peroxide (H 2 O 2 ), and in the detection step horseradish peroxidase uses H 2 O 2 to convert the colorless, nonfluorescent reagent Amplex Red to the red-colored and highly fluorescent product resorufin. The assay was validated in both the absorbance and the fluorescence modes. We show that, due to its high sensitivity and substrate selectivity, this assay can be used to measure enzymatic activity in human serum containing L-asparaginase.Ó 2013 Elsevier Inc. All rights reserved.The enzyme L-asparaginase (EC 3.5.1.1, L-asparagine amidohydrolase, L-ASNase), 1 which predominantly occurs in microorganisms and plants, catalyzes the hydrolysis of L-asparagine (L-Asn) to L-aspartic acid (L-Asp) and ammonia [1,2]. Escherichia coli L-ASNase has been used extensively as a therapeutic enzyme in the frontline treatment of lymphoblastic malignancies, such as acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma [3,4], since the 1960s. In light of the significance of L-ASNase as a therapeutic protein, various methods have been developed for measuring the enzyme's activity. Those assays can be used in either absorbance mode [5][6][7][8][9][10] or fluorescence mode [11,12]. However, these assays suffer from certain disadvantages that are primarily related to the use of substrate analogs instead of the natural substrate L-Asn [6,7,11,12]. Such assays are not suitable for in vitro evolution of L-asparaginases that aims to select variants showing improved catalytic efficiency and selectivity for the physiological substrate. Moreover, the limited sensitivity of absorption-based spectrophotometric assays [5][6][7]9,10] is a major handicap to significantly reducing reaction volumes. A noteworthy example of such a case is droplet-based microfluidics, which has emerged as a powerful tool for high-throughput screening in directed protein evolution [13,14]. In these experimental setups, the assay volume is minimized to 1 nl, to 1 fl, scaling down light path lengths to 1 lm.Here, we report on the development of a novel L-ASNase assay that can be used in either the fluorescence or absorbance mode and relies solely on the use of the physiological substrate L-Asn. In this three-step coupled enzyme system (Fig. 1), L-Asp, which is one of the two products of the L-ASNase reaction, is oxidized by L-aspartate oxidase (L-AspOx), resulting in the formation of iminoaspartate and hydrogen peroxide (H 2 O 2 ); the latter product is used by horseradish peroxidase (HRP) to oxidize the nonfluorescent compound Amplex Red (AR) to resorufin, which exhibits excellent fluorescence as well as absorbance properties.For establishing and quantitatively evaluating the assay, we cloned and recombinantly produced E. coli L-AspOx [15]...

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A sensitive fluorimetric assay for pyruvate

Cited by 63 publications

References 22 publications

L-2-Hydroxyglutarate production arises from noncanonical enzyme function at acidic pH

L-2-Hydroxyglutarate production arises from noncanonical enzyme function at acidic pH

Increased Glycolytic ATP Synthesis Is Associated with Tafenoquine Resistance inLeishmania major

An Amplex Red-based fluorometric and spectrophotometric assay for l-asparaginase using its natural substrate

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