Measurement of Enzyme Activities and Optimization of Continuous and Discontinuous Assays

Bénard, Camille; Gibon, Yves

doi:10.1002/cppb.20003

Cited by 10 publications

(7 citation statements)

References 18 publications

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“…The initial rates were consistently higher (beyond Lane C) in the proposed protocol ( Figure S3 ) and the difference between them was significant after a post-hoc pairwise t -test ( Table 1 ). In brief, the split of the assay buffer into two halves is advantageous when the catalase activity is measured simultaneously in several samples and there are no other advanced technical alternatives to speed up the buffer loading with pipetting robots [ 16 , 30 ].…”

Section: Resultsmentioning

confidence: 99%

Theoretical and Experimental Considerations for a Rapid and High Throughput Measurement of Catalase In Vitro

et al. 2021

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A rapid and high throughput protocol to measure the catalase activity in vitro has been designed. Catalase is an enzyme with unusual kinetic properties because it does not follow the standard Michaelis–Menten model and is inactivated by H2O2. This makes the analysis of the two rate equations of the second-ordered reactions of the kinetic model rather complex. A two-degree polynomial fitting of the experimental data is proposed after transforming the exponential form of the integrated rate equation of the [H2O2] into a polynomial using the Taylor series. The fitting is validated by establishing an experimental linear relationship between the initial rate of the H2O2 decomposition and the protein concentration, regardless of the suicide inactivation that catalase might undergo beyond t > 0. In addition, experimental considerations are taken into account to avoid statistical bias in the analysis of the catalase activity. ANOVA analyses show that the proposed protocol can be utilized to measure the initial rate of the H2O2 decomposition by catalase in 32 samples in triplicates if kept below 8 mM min−1 in the microplate wells. These kinetic and statistical analyses can pave the way for other antioxidant enzyme activity assays in microplate readers at small scale and low cost.

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Section: Resultsmentioning

confidence: 99%

Theoretical and Experimental Considerations for a Rapid and High Throughput Measurement of Catalase In Vitro

et al. 2021

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“…Stem discs were stored at −80°C before grinding into a fine powder in liquid nitrogen with a mortar and pestle. 20 mg of woody tissue material was used for the discontinuous assay performed in a 96‐well microplate (Bénard & Gibon, 2016). Briefly, aliquots were extracted by shaking with an extraction buffer.…”

Section: Methodsmentioning

confidence: 99%

Differences between tree stem CO₂ efflux and O₂ influx rates cannot be explained by internal CO₂ transport or storage in large beech trees

Helm

Salomón

Hilman

et al. 2023

Plant Cell & Environment

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Tree stem respiration (RS) is a substantial component of the forest carbon balance. The mass balance approach uses stem CO2 efflux and internal xylem fluxes to sum up RS, while the oxygen‐based method assumes O2 influx as a proxy of RS. So far, both approaches have yielded inconsistent results regarding the fate of respired CO2 in tree stems, a major challenge for quantifying forest carbon dynamics. We collected a data set of CO2 efflux, O2 influx, xylem CO2 concentration, sap flow, sap pH, stem temperature, nonstructural carbohydrates concentration and potential phosphoenolpyruvate carboxylase (PEPC) capacity on mature beech trees to identify the sources of differences between approaches. The ratio of CO2 efflux to O2 influx was consistently below unity (0.7) along a 3‐m vertical gradient, but internal fluxes did not bridge the gap between influx and efflux, nor did we find evidence for changes in respiratory substrate use. PEPC capacity was comparable with that previously reported in green current‐year twigs. Although we could not reconcile differences between approaches, results shed light on the uncertain fate of CO2 respired by parenchyma cells across the sapwood. Unexpected high values of PEPC capacity highlight its potential relevance as a mechanism of local CO2 removal, which merits further research.

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“…All assays were performed on microplates using 96-head pipetting robots (Hamilton, Villebon-sur-Yvette, France) and UV-visible readers (SAFAS, Monaco, France). Assay optimization and calculations were conducted as described in Bénard and Gibon (2016) .…”

Section: Methodsmentioning

confidence: 99%

Overexpression of thioredoxin m in chloroplasts alters carbon and nitrogen partitioning in tobacco

Ancín

Larraya

Florez‐Sarasa

et al. 2021

Journal of Experimental Botany

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In plants, there is a complex interaction between carbon (C) and nitrogen (N) metabolism and its coordination is fundamental for plant growth and development. In the present work, the influence of thioredoxin (Trx) m on C and N partitioning was studied using tobacco plants overexpressing Trx m from the chloroplast genome. The transgenic plants showed altered metabolism of C (lower leaf starch and soluble sugar accumulation) and N (with higher amounts of amino acids and soluble protein), which pointed to an activation of N metabolism at the expense of carbohydrates. To further delineate the effect of Trx m overexpression, metabolomic and enzymatic analyses were performed on these plants, indicating an up-regulation of the GS-GOGAT pathway. Trx m-overexpressing plants specifically displayed increased activity and stability of glutamine synthetase in tobacco plants. Moreover, higher photorespiration and nitrate accumulation were determined in these plants relative to the untransformed control, indicating that overexpression of Trx m favors the photorespiratory N cycle rather than primary nitrate assimilation. Taken together, the combined results reveal the importance of Trx m as a molecular mediator of N metabolism in plant chloroplasts.

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Measurement of Enzyme Activities and Optimization of Continuous and Discontinuous Assays

Cited by 10 publications

References 18 publications

Theoretical and Experimental Considerations for a Rapid and High Throughput Measurement of Catalase In Vitro

Theoretical and Experimental Considerations for a Rapid and High Throughput Measurement of Catalase In Vitro

Differences between tree stem CO₂ efflux and O₂ influx rates cannot be explained by internal CO₂ transport or storage in large beech trees

Overexpression of thioredoxin m in chloroplasts alters carbon and nitrogen partitioning in tobacco

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Measurement of Enzyme Activities and Optimization of Continuous and Discontinuous Assays

Cited by 10 publications

References 18 publications

Theoretical and Experimental Considerations for a Rapid and High Throughput Measurement of Catalase In Vitro

Theoretical and Experimental Considerations for a Rapid and High Throughput Measurement of Catalase In Vitro

Differences between tree stem CO2 efflux and O2 influx rates cannot be explained by internal CO2 transport or storage in large beech trees

Overexpression of thioredoxin m in chloroplasts alters carbon and nitrogen partitioning in tobacco

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Differences between tree stem CO₂ efflux and O₂ influx rates cannot be explained by internal CO₂ transport or storage in large beech trees