Capillary electrophoresis-based assay of phosphofructokinase-1

Malina, Andrew; Bryant, Sherrisse; Chang, Sheng-Fuh; Waldrop, Grover L.; Gilman, S. Douglass

doi:10.1016/j.ab.2013.10.028

Cited by 7 publications

(9 citation statements)

References 36 publications

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“…Peak splitting of ADP and ATP was eliminated, similar to the findings of Malina et al. , and a peak overlapping with malonyl‐CoA was eliminated. The remaining Mg‐ATP and Mg‐ADP peaks migrating before acetyl‐CoA were better resolved with Mg 2+ .…”

Section: Resultssupporting

confidence: 80%

“…The ACC2 assay based on CE depends on the separation and quantification of the nucleotide substrates and products – ATP, ADP, acetyl‐CoA, and malonyl‐CoA. Previous CE assays for bacterial ACC and phosphofructokinase‐1 included methods for separation of nucleotides based on earlier published separations ; however, the development of the ACC2 assay inspired a re‐examination of those previous nucleotide separations.…”

Section: Resultsmentioning

confidence: 99%

“…Separation of these four molecules has been demonstrated for an assay of bacterial ACC , but optimum solution conditions for human ACC2 and bacterial ACC are significantly different . The separation was revisited, based on the reaction requirements of human ACC2 and our experience working with bacterial ACC and phosphofructokinase‐1 (PFK‐1), another Mg 2+ ‐dependent enzyme . Figure shows an electropherogram obtained using conditions developed for the bacterial holo‐ACC enzyme assay where the sample buffer was 5.0 mM potassium phosphate, 2.5 mM MgCl 2 , and 5.0 mM KHCO 3 at pH 7.50, and the separation buffer was 10.0 mM sodium phosphate and 20.0 mM SDS at pH 7.50 .…”

Section: Resultsmentioning

confidence: 99%

“…Later work by Malina, et al. on the development of a CE assay for PFK‐1 shed light on the issue . The substrate for both ACC and PFK‐1 is ATP complexed to Mg 2+ , so Mg 2+ must be included in the reaction solution, which is injected into the CE capillary for the enzyme assays.…”

Section: Resultsmentioning

confidence: 99%

“…Separation reproducibility of ATP and ADP for the PFK‐1 assay was improved markedly by adding 1 mM Mg 2+ to the separation buffer. The peaks in the CE separations were Mg‐ATP and Mg‐ADP complexes, and additional peaks in the electropherograms with poor reproducibility were attributed to dissociation of Mg‐ATP and Mg‐ADP during the CE separations without Mg 2+ in the separation buffer .…”

Section: Resultsmentioning

confidence: 99%

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Capillary electrophoretic assay of human acetyl‐coenzyme A carboxylase 2

2019

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Human acetyl‐coenzyme A carboxylase 2 catalyzes the carboxylation of acetyl coenzyme A to form malonyl coenzyme A, along with the conversion of magnesium‐adenosine triphosphate complex to magnesium‐adenosine diphosphate complex. A simple off‐column capillary electrophoresis assay for human acetyl‐coenzyme A carboxylase 2 was developed based on the separation of magnesium‐adenosine triphosphate complex, magnesium‐adenosine diphosphate complex, acetyl coenzyme A and malonyl coenzyme A with detection by ultraviolet absorption at 256 nm. When Mg2+ was absent from the separation buffer, the zones due to magnesium‐adenosine triphosphate complex and magnesium‐adenosine diphosphate complex both split and migrated as two separate peaks. With Mg2+ added to the separation buffer, magnesium‐adenosine triphosphate complex and magnesium‐adenosine diphosphate complex produced single peaks, and the reproducibility of peak shape and area improved for human acetyl‐coenzyme A carboxylase 2 assay components. The final separation buffer used was 30.0 mM HEPES, 3.0 mM MgCl2, 2.5 mM KHCO3, and 2.5 mM potassium citrate at pH 7.50. The same buffer was used for the enzyme‐catalyzed reaction (off‐column). Inhibition of human acetyl‐coenzyme A carboxylase 2 by CP‐640186, a known inhibitor, was detected using the capillary electrophoresis assay.

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Section: Resultssupporting

confidence: 80%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Capillary electrophoretic assay of human acetyl‐coenzyme A carboxylase 2

2019

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Advances in Capillary Electrophoresis-Based Enzyme Assays

Scriba

Belal

2015

Chromatographia

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IntroductionEnzymes catalyze metabolic reactions in cells regulating homeostasis, growth and reproduction of living organisms. Enzymes are also often involved in human disease. The analysis of the human genome has revealed that within the so-called druggable genome a significant portion (in some analyses more than half) of the potential drug targets are enzymes [1][2][3]. Consequently, enzymes are important targets in the development of new drugs. Moreover, enzymes play a role in the clinical diagnosis of diseases. Therefore, effective methods for characterization of enzymes as well as for the determination of their activity are important, for example, for the understanding of enzyme-mediated metabolic reactions or the identification of substrates and inhibitors for the treatment of human diseases.Capillary electrophoresis (CE) has been applied to enzyme analysis for more than 20 years since the first report by Banke et al. on an assay of alkaline protease [4]. Since then, all aspects of enzyme-related analysis have been studied by CE methods including the evaluation of enzymatic activity, enzyme kinetics, identification and characterization of enzyme inhibitors and activators as well as metabolic pathways as, for example, summarized in [5][6][7][8][9][10][11][12].Generally, CE-based enzyme assays can be divided in three groups, pre-capillary (offline) assays, in-capillary (online) assays and post-capillary assays. In the pre-capillary assays, all required components including enzyme, substrate, co-factors, etc., are mixed in a vial and incubated for an intended period of time. Subsequently, the reaction is quenched and an aliquot is subjected to CE analysis for the determination of substrate(s) and/or co-substrate(s) and/ or product(s). Multiple sampling or repeated sampling in combination with sequential runs for the determination of Abstract Capillary electrophoresis (CE) has become a flexible and accurate, high-efficiency analytical separation technique in many areas requiring only minute amounts of sample and chemicals. Thus, CE has also been recognized as a suitable technique to study enzymatic reactions including the determination of Michaelis-Menten kinetic data or the identification and characterization of inhibitors. The most often applied CE-based enzyme assay modes can be divided into two categories: (1) pre-capillary assays where incubations are performed offline followed by CE analysis of substrate(s) and/or product(s) and (2) in-capillary assays in which the enzymatic reaction and analyte separation are performed in the same capillary. In case of the in-capillary assays, the enzyme may be immobilized or in solution. The latter is also referred to as electrophoretically mediated microanalysis (EMMA), while in the case of immobilized enzyme the term immobilized enzyme reactor (IMER) is used. The present review summarizes the literature on CEbased enzyme assays published between January 2010 and April 2015. Immobilized enzyme reactors as well as microfluidic devices applied to the study of enzymatic a...

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Recent advances in screening of enzymes inhibitors based on capillary electrophoresis

Cheng

Chen

2018

Journal of Pharmaceutical Analysis

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Capillary electrophoresis with many advantages plays an important role in pharmaceutical analysis and drug screening. This review gives an overview on the recent advances in the developments and applications of capillary electrophoresis in the field of enzyme inhibitor screening. The period covers 2013 to 2017. Both the pre-capillary enzyme assays and in-capillary enzyme assays which include electrophoretically mediated microanalysis (EMMA) and immobilized enzyme microreactor (IMER) are summarized in this article.

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Capillary electrophoresis-based assay of phosphofructokinase-1

Cited by 7 publications

References 36 publications

Capillary electrophoretic assay of human acetyl‐coenzyme A carboxylase 2

Capillary electrophoretic assay of human acetyl‐coenzyme A carboxylase 2

Advances in Capillary Electrophoresis-Based Enzyme Assays

Recent advances in screening of enzymes inhibitors based on capillary electrophoresis

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