A capillary electrophoretic assay for acetyl coenzyme A carboxylase

Bryant, Sherrisse; Waldrop, Grover L.; Gilman, S. Douglass

doi:10.1016/j.ab.2013.02.005

Cited by 8 publications

(12 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The first separation buffer used during the development of this assay contained 15.0 mM Tris-HCl and 30 mM SDS at pH 8.00. It has been reported that addition of SDS improves the separation of ATP and ADP [20; 21]. Under these conditions (above the SDS critical micelle concentration), the separation is a micellar enhanced capillary electrokinetic chromatography (MEKC) separation [22].…”

Section: Resultsmentioning

confidence: 99%

Capillary electrophoresis-based assay of phosphofructokinase-1

Malina

Bryant

Chang

et al. 2014

Analytical Biochemistry

Self Cite

View full text Add to dashboard Cite

An assay was developed for phosphofructokinase-1 (PFK-1) using capillary electrophoresis (CE). In the glycolytic pathway, this enzyme catalyzes the rate-limiting step from fructose-6-phosphate and magnesium-bound adenosine triphosphate (Mg-ATP) to fructose-1,6-bisphosphate and magnesium-bound adenosine diphosphate (Mg-ADP). This enzyme has recently become a research target because of the importance of glycolysis in cancer and obesity. The CE assay for PFK-1 is based on the separation and detection by UV absorbance at 260 nm of Mg-ATP and Mg-ADP. The separation was enhanced by addition of Mg2+ to the separation buffer. Inhibition studies of PFK-1 by aurintricarboxylic acid and palmitoyl coenzyme A were also performed. An IC50 value was determined for aurintricarboxylic acid, and this value matched values in the literature obtained using coupled spectrophotometric assays. This assay for PFK-1 directly monitors the enzyme-catalyzed reaction, and the CE separation reduces the potential of spectral interference by inhibitors.

show abstract

Section: Resultsmentioning

confidence: 99%

Capillary electrophoresis-based assay of phosphofructokinase-1

Malina

Bryant

Chang

et al. 2014

Analytical Biochemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…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%

“…The injected sample will contain the solution used for the enzyme‐catalyzed reaction, and its injection must not degrade the CE separation. 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 .…”

Section: Resultsmentioning

confidence: 99%

“…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 . These conditions have been used successfully for quantitative assays of bacterial holo‐ACC and its inhibition, but this method occasionally suffered from reproducibility problems and peak splitting for ADP and ATP (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Electrophoretic separation of substrates, products, and inhibitors in CE‐based assays minimizes the potential for spectral interference from inhibitors. Recently, a CE enzyme assay for ACC from E. coli was developed . This assay can be used for holo‐ACC‐catalyzed reactions as well as the half‐reactions catalyzed by the isolated BC and CT components.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Capillary electrophoretic assay of human acetyl‐coenzyme A carboxylase 2

2019

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Advances in Capillary Electrophoresis-Based Enzyme Assays

Scriba

Belal

2015

Chromatographia

View full text Add to dashboard Cite

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...

show abstract

A capillary electrophoretic assay for acetyl coenzyme A carboxylase

Cited by 8 publications

References 33 publications

Capillary electrophoresis-based assay of phosphofructokinase-1

Capillary electrophoresis-based assay of phosphofructokinase-1

Capillary electrophoretic assay of human acetyl‐coenzyme A carboxylase 2

Advances in Capillary Electrophoresis-Based Enzyme Assays

Contact Info

Product

Resources

About