Techniques for Measuring the Activity of Carboxylic Acid:CoA Ligase and Acyl‐CoA:Amino Acid N‐Acyltransferase: The Amino Acid Conjugation Pathway
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- Abstract
- Table of Contents
- Materials
- Figures
- Literature Cited
Abstract
A wide variety of xenobiotic carboxylic acids are metabolized to their amino acid conjugates via a pathway that exists primarily in liver and kidney. This conjugation occurs in a two?step pathway catalyzed by two distince types of enzymes, ligases and transferases. Measurements of acyl?CoA ligase activity include monitoring the rate of appearance of AMP or PPi, or the CoA adduct. N?acyltransferases catalyze formation of an amino acid conjugate from the CoA?activated intermediate, releasing CoA. This reaction is monitored by following the release of free CoA or the disappearance of the acyl?CoA adduct.
Table of Contents
- Basic Protocol 1: Direct Measurement of Carboxylic Acid:CoA Ligase Activity using a Radiolabeled Carboxylic Acid
- Alternate Protocol 1: Measurement of Radiolabeled ATP Cleavage by Carboxylic Acid:CoA Ligase
- Alternate Protocol 2: Spectrophotometric Assay of Carboxylic Acid:CoA Ligase Activity using DTA
- Alternate Protocol 3: Indirect Assay of Carboxylic Acid:CoA Ligase using AMP Formation Coupled to NADH Oxidation
- Alternate Protocol 4: Monitoring Carboxylic Acid:CoA Ligase Activity by HPLC Determination of Acyl‐CoA Product Formation
- Basic Protocol 2: Monitoring CoA Release using a DTNB‐coupled Assay to Determine N‐Acylasetransferase Activity
- Alternate Protocol 5: Direct Assay of N‐Acyltransferase Activity by Monitoring the Disappearance of Thioester Bond Absorbance
- Alternate Protocol 6: Radioactive Assays for N‐Acyltransferases
- Alternate Protocol 7: HPLC Assays for N‐Acyltransferases
- Reagents and Solutions
- Commentary
- Literature Cited
- Figures
- Tables
Materials
Basic Protocol 1: Direct Measurement of Carboxylic Acid:CoA Ligase Activity using a Radiolabeled Carboxylic Acid
Materials
Alternate Protocol 1: Measurement of Radiolabeled ATP Cleavage by Carboxylic Acid:CoA Ligase
Alternate Protocol 2: Spectrophotometric Assay of Carboxylic Acid:CoA Ligase Activity using DTA
Alternate Protocol 3: Indirect Assay of Carboxylic Acid:CoA Ligase using AMP Formation Coupled to NADH Oxidation
Materials
Alternate Protocol 4: Monitoring Carboxylic Acid:CoA Ligase Activity by HPLC Determination of Acyl‐CoA Product Formation
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Figures
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Figure 4.11.1 (A ) Data from the scintillation counter are plotted as counts per minute (cpm) versus reaction time using 300 µM ATP and [14 C]benzoate concentrations of 1.2 (open circles), 2 (closed circles), 4 (open triangles), 10 (closed triangles), and 20 µM (open squares). Rates (uncorrected for extraction loss) are calculated from these data, which represent one‐tenth of the total reaction, using the formula: [radioactivity (cpm)/incubation time (min) × dilution factor]/specific activity of substrate (cpm/pmol) = amount acylated product (pmol)/min/volume enzyme = V . (B ) Reciprocals of these rates (1/ V ) are plotted versus the reciprocal of the benzoate concentration for these 300 µM ATP rates (open squares), as well as for rates conducted at ATP concentrations of 15 µM (open circles), 20 µM (closed circles), 29 µM (open triangles), and 50 µM (closed triangles). View Image -
Figure 4.11.2 Data from the scintillation counter are plotted as cpm (divided by 1 × 103 for convenience) versus reaction time for the substrates hydrocinnamic acid (open triangles) and indole‐3‐propionic acid (closed circles), as well as a control reaction omitting CoA (open circles). Rates are calculated from these data, which represent one‐tenth of the total reaction using the following formula: [radioactivity (cpm)/incubation time (min) × dilution factor]/specific activity of ATP (cpm/pmol) = amount inorganic phosphate formed (pmol)/min/volume enzyme = V . View Image -
Figure 4.11.3 Lineweaver‐Burk double reciprocal plots of the rates of salicyl‐CoA glycine conjugation (as nanomoles per minute per milliliter enzyme) versus the concentration of salicyl‐CoA for four different glycine concentrations: 5 (open circles), 10 (closed circles), 20 (open triangles), and 50 mM (closed triangles). Rates are calculated from the absorbance change with time using the extinction coefficient for DTNB: (ΔOD412 /incubation time (min)/volume enzyme)/[(13.6 AU liters/mmol) × 1000 ml/liter] = amount product formed (mmol)/min/100 µl enzyme = V . View Image
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Literature Cited
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