Techniques for Measuring the Activity of Carboxylic Acid:CoA Ligase and Acyl‐CoA:Amino Acid N‐Acyltransferase: The Amino Acid Co实验方法详情页

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Techniques for Measuring the Activity of Carboxylic Acid:CoA Ligase and Acyl‐CoA:Amino Acid N‐Acyltransferase: The Amino Acid Co

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Abstract
Table of Contents
Materials
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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.

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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

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Basic Protocol 1: Direct Measurement of Carboxylic Acid:CoA Ligase Activity using a Radiolabeled Carboxylic Acid Materials

Kill/acidification mix (see recipe ) in dispenser set to deliver 0.4 ml
Water‐saturated butanol (see recipe ) in dispenser set to deliver 1 ml
1 M Tris⋅Cl, pH 8.0 at 30°C ( appendix 2A )
1 M KCl
100 mM MgCl 2
5 mM coenzyme A (see recipe )
Radiolabeled carboxylic acid substrate (e.g., 100 µM 80 cpm/mol [14 C]benzoate; see recipe )
1 mM ATP (see recipe )
Carboxylic acid:CoA ligase preparation (see )
Scintillation fluid

Positive‐displacement pipet (e.g., Drummond)
30°C water bath
Vacuum pipettor
Scintillation vials

CAUTION: When working with radioactivity, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in an appropriately designated area, following the guidelines provided by the local radiation safety officer (also see appendix 1A ). Also note that short‐chain fatty acids are volatile and should be maintained in sealed tubes to avoid contamination of storage areas. Alternate Protocol 1: Measurement of Radiolabeled ATP Cleavage by Carboxylic Acid:CoA Ligase

ATP‐assay kill mix (50 mM Na 2 EDTA/5 mM K 2 HPO 4 ) in a dispenser set to deliver 1.9 ml
Unlabeled carboxylic acid substrate (e.g., 1 mM hydrocinnamic acid)
[γ‐33 P]ATP of known concentration and specific activity (e.g., 10 mM, 6.8 cpm/pmol; also see recipe for radiolabeled carboxylic acid substrates for specific activity determination)
Phosphoric and sulfuric acid‐washed activated charcoal (e.g., Sigma)

CAUTION: When working with radioactivity, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in an appropriately designated area, following the guidelines provided by the local radiation safety officer (also see appendix 1A ). Alternate Protocol 2: Spectrophotometric Assay of Carboxylic Acid:CoA Ligase Activity using DTA

5 mM DTA (see recipe )
Recording spectrophotometer, thermostatically‐controlled at 30°C

Alternate Protocol 3: Indirect Assay of Carboxylic Acid:CoA Ligase using AMP Formation Coupled to NADH Oxidation Materials

1 mM DTNB (see recipe )
1 M KCl
1 mM benzoyl‐CoA
N ‐acyltransferase preparation (see )
Acceptor amino acid (i.e., glycine or glutamine)
Recording spectrophotometer, thermostatically‐controlled at 30°C

Alternate Protocol 4: Monitoring Carboxylic Acid:CoA Ligase Activity by HPLC Determination of Acyl‐CoA Product Formation

0.5 M Tris⋅Cl, pH 8.0 at 30°C ( appendix 2A )
1‐ml quartz cuvettes

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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).

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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 .

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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 .

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Literature Cited

Literature Cited
	Bronfman, M., Amigo, L., and Morales, M.N. 1986. Activation hypolipidaemic drugs to acylcoenzyme A thioesters. Biochem. J. 239:781‐784.
	Brugger, R., Alia, B.G., Reichel, C., Waibel, R., Menzel, S., Brune, K., and Geisslinger, G. 1996. Isolation and characterization of rat liver microsomal R‐ibuprofenoyl‐CoA synthethase. Biochem. Pharmacol. 52:1007‐1013.
	Dawson, A., Elliott, D.C., Elliott, W.H., and Jones, K.M. 1986. Data for Biochemical Research, Third Edition. Clarendon Press, Oxford.
	Garland, P.B., Yates, D.W., and Haddock, B.A. 1970. Spectrophotometric studies of acyl‐coenzyme A synthetases of rat liver mitochondria. Biochem. J. 119:553‐564.
	Gregus, Z., Fekete, T., Halaszi, E., and Klaassen, C.D. 1996. Lipoic acid impairs glycine conjugation of benzoic acid and renal excretion of benzoylglycine. Drug Metab. Disp. 24:682‐688
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	Kasuya, F., Igarashi, K., and Fukui, M. 1990. Glycine conjugation of the substituted benzoic acids in vitro: Structure‐metabolism relationship study. J. Pharmacobio. Dyn. 13:432‐440.
	Kasuya, F., Igarashi, K., Fukui, M., and Nokihara, K. 1996. Purification and characterization of a medium chain acyl‐coenzyme A synthetase. Drug Metab. Dispos. 24:879‐883.
	Kelley, M. and Vessey, D.A. 1990. The effects of ions on the conjugation of xenobiotics by the aralkyl‐CoA and arylacetyl‐CoA N‐acyltransferases from bovine liver mitochondria. J. Biochem. Tox. 5:125‐135.
	Knights, K.M. and Roberts, B.J. 1994. Xenobiotic acyl‐CoA formation: Evidence of kinetically distinct hepatic microsomal long‐chain fatty acid and nafenopin‐CoA ligases. Chemico‐Biol. Interact. 90:215‐223.
	Rodriguez‐Aparicio, L.B., Reglero, A., Martinex‐Blanco, H., and Luengo, J.M. 1991. Fluorimetric determination of phenylacetylCoA ligase from Pseudomonas putida: A very sensitive assay for a newly described enzyme. Biochim. Biophys Acta 1073:431‐433.
	Vessey, D.A. 1997. Enzymes involved in the formation of amide bonds. In Comprehensive Toxicology Vol. 3, Biotransformation (Guengerich, F.P., ed.), p. 455‐475. Elsevier Science, Oxford.
	Vessey, D.A., Crissey, M.H., and Zakim, D. 1977. Kinetic studies on the enzymes conjugating bile acids with taurine and glycine in bovine liver. Biochem. J. 163:181‐183.
	Webster, L.T. Jr., Siddiqui, U.A., Lucas, S.V., Strong, J.M., and Mieyal, J.J. 1976. Identification of separate acyl‐CoA:glycine and acyl‐CoA:glutamine N‐acyltransferase activities in mitochondrial fractions from liver of rhesus monkey and man. J. Biol. Chem. 251:3352‐3358.

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