Enzymatic Activity of Human Carboxylesterases

Matthew K. Ross1, Abdolsamad Borazjani1

1 Mississippi State University, Mississippi State, Mississippi
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 4.24
DOI:  10.1002/0471140856.tx0424s33
Online Posting Date:  August, 2007
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Abstract

The carboxylesterases (CEs) are hydrolytic enzymes that metabolize xenobiotics that contain ester, thioester, or amide bonds. CEs are ubiquitously expressed but are found in highest concentration in membrane‐enriched fractions of the liver. This unit describes assays used to measure the enzymatic activity and tissue distribution of human CEs. Both spectrophotometric and HPLC‐based assays are described in detail. Several of these methods are rapid and easy to perform. These methods will be useful to investigators who are interested in studying the metabolism and disposition of esterified drugs and environmental chemicals. Curr. Protoc. Toxicol. 33:4.24.1‐4.24.14. © 2007 by John Wiley & Sons, Inc.

Keywords: carboxylesterases; hydrolysis; pesticides; xenobiotic metabolism; liver

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Continuous Spectrophotometric Assay to Measure Carboxylesterase Activity
  • Alternate Protocol 1: Continuous Spectrofluorometric Assay to Measure Carboxylesterase Activity
  • Basic Protocol 2: Discontinuous HPLC‐Based Assay to Measure Carboxylesterase Activity Using Purified Enzyme
  • Alternate Protocol 2: Discontinuous HPLC‐Based Assay to Measure Carboxylesterase Activity Using Microsomes
  • Support Protocol 1: HPLC Analysis of Pyrethroid Hydrolysis Products
  • Alternate Protocol 3: HPLC‐Based Assays of Procaine Hydrolysis
  • Support Protocol 2: Kinetic Analysis and Statistics
  • Basic Protocol 3: Native In‐Gel Hydrolysis Assay Measurement of Human Carboxylesterases
  • Basic Protocol 4: SDS‐PAGE of Human Liver Microsomes and Western Blotting of Carboxylesterases 1 and 2
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Continuous Spectrophotometric Assay to Measure Carboxylesterase Activity

  Materials
  • 100 mM p‐nitrophenyl valerate (Sigma‐Aldrich) in ethanol (store up to 3 months at −20°C) or 100 mM p‐nitrophenyl acetate (Sigma‐Aldrich) in ethanol (store up to 3 months at −20°C)
  • 50 mM Tris⋅Cl buffer, pH 7.4
  • Human or rodent liver subcellular fractions (microsomes and cytosols prepared as described by Guengerich, ) or pure carboxylesterase proteins (rodent or human)—recombinantly expressed or purified from liver sources (see Morton and Potter, , for methods to prepare recombinant CEs)
  • Corning clear 96‐well plates (Fisher Scientific)
  • Plate reader (ThermoMax visible wavelength plate reader, fMax fluorescent plate reader, Molecular Devices)
NOTE: Most protocols for preparing tissue subcellular fractions (i.e., microsomes and cytosols) will be compatible with the assays to be described below. However, it is essential that no protease inhibitors (e.g., phenyl methylsulfonyl fluoride, PMSF) are added to homogenization buffers since these compounds are mechanism‐based inhibitors that will also inhibit CEs, in addition to the serine proteases that they are intended for, e.g., trypsin.

Alternate Protocol 1: Continuous Spectrofluorometric Assay to Measure Carboxylesterase Activity

  • 100 mM 4‐methylumbelliferyl acetate (4‐MUBA; Sigma‐Aldrich) in acetonitrile (store up to 3 months at −20°C)
  • 100 mM 4‐methylumbelliferone (4‐MUB; Sigma‐Adrich) in acetonitrile (store up to 3 months at −20°C)
  • Corning 96‐well black plates with clear bottoms (Fisher Scientific)
NOTE: Most protocols for preparing tissue subcellular fractions (i.e., microsomes and cytosols) will be compatible with the assays to be described below. However, it is essential that no protease inhibitors (e.g., phenyl methylsulfonyl fluoride, PMSF) are added to homogenization buffers since these compounds are mechanism‐based inhibitors that will also inhibit CEs, in addition to the serine proteases that they are intended for, e.g., trypsin.

Basic Protocol 2: Discontinuous HPLC‐Based Assay to Measure Carboxylesterase Activity Using Purified Enzyme

  Materials
  • Pyrethroid substrates (e.g., permethrin, bioresmethrin, and deltamethrin; Chem Service)
  • Acetonitrile, HPLC grade and ice cold
  • 50 mM Tris⋅Cl buffer, pH 7.4
  • Pure carboxylesterase proteins (rodent or human)—recombinant or purified from liver
  • 3‐(4‐Methoxy)‐phenoxybenzaldehyde (Sigma‐Aldrich)
  • 37°C incubator with shaker
  • HPLC chromatograph

Alternate Protocol 2: Discontinuous HPLC‐Based Assay to Measure Carboxylesterase Activity Using Microsomes

  • Pyethroid substrate
  • Human or rodent liver subcellular fractions (microsomes and cytosols)
  • Tetraethylpyrophosphate (Chem Service)

Support Protocol 1: HPLC Analysis of Pyrethroid Hydrolysis Products

  Materials
  • Solvent A: 1:1 (v/v) water/acetonitrile containing 0.1% (v/v) acetic acid
  • Solvent B: 100% acetonitrile containing 0.1% (v/v) acetic acid
  • Hydrolysis products of pyrethroid (see protocol 3 or protocol 4)
  • 3‐(4‐methoxy)‐phenoxybenzaldehyde
  • Vacuum
  • Sonicator
  • Reversed‐phase C18 HPLC column, 2.1 × 100–mm (Thermo Electron)
  • Surveyor LC system (Thermo Electron) or Alliance LC system (Waters)
  • UV detector

Alternate Protocol 3: HPLC‐Based Assays of Procaine Hydrolysis

  • 10 mM procaine (Sigma‐Aldrich) in ethanol
  • Liver microsomes (human)
  • 300 µM 3‐aminobenzoic acid (internal standard; Sigma‐Aldrich) in acetonitrile
  • 0.5‐ml microcentrifuge tubes
  • 37°C incubator (e.g., Eppendorf Thermomixer or equivalent)

Support Protocol 2: Kinetic Analysis and Statistics

  Materials
  • Individual and pooled human liver microsomes (commercially purchased, e.g., BD Biosciences, or prepared in the laboratory)
  • 2× native loading buffer (Bio‐Rad or see recipe)
  • 10% native polyacrylamide mini‐gels (10% resolving gel with a 4% stacking gel; Bio‐Rad)
  • 100 mM 4‐methylumbelliferyl acetate (4‐MUBA; Sigma‐Adrich) in acetonitrile (store up to 3 months at −20°C)
  • 100 mM potassium phosphate buffer, pH 6.5
  • Gel apparatus
  • Platform rocker
  • UV transilluminator plate
  • Gel documentation system (Alpha Inotech)

Basic Protocol 3: Native In‐Gel Hydrolysis Assay Measurement of Human Carboxylesterases

  Materials
  • Individual and pooled human liver microsomes (purchased commercially or prepared in the laboratory)
  • 2× SDS‐loading buffer (see recipe)
  • 10% SDS‐PAGE gel
  • TBS (see recipe) containing 0.1% (v/v) Tween 20 and 5% (w/v) nonfat dry milk (at 4°C)
  • Primary antibody: polyclonal rabbit antibodies raised against hCE1 and hCE2 (rabbit anti‐hCE1 or anti‐hCE2)
  • Secondary antibody: Goat anti‐rabbit horseradish peroxidase‐conjugated IgG
  • Supersignal West Pico chemiluminescent substrate (Pierce)
  • Recombinant human carboxylesterases 1 and 2 (for preparation, see Morton and Potter, )
  • Gel apparatus
  • Transfer apparatus
  • Polyvinylidene difluoride (PVDF) membranes
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Figures

Videos

Literature Cited

Literature Cited
   Crow, J.A., Borazjani, A., Potter, P.M., and Ross, M.K. 2007. Hydrolysis of pyrethroids by human and rat tissue: Examination of intestinal, liver, and serum carboxylesterases. Toxicol. Appl. Pharmacol. 221:1‐12.
   Dean, R.A., Zhang, J., Brzezinski, M.R., and Bosron, W.F. 1995. Tissue distribution of cocaine methyl esterase and ethyl transferase activities: Correlation with carboxylesterase protein. J. Pharmacol. Exp. Ther. 275:965‐971.
   Dolinsky, V.W., Gilham, D., Alam, M., Vance, D.E., and Lehner, R. 2004. Triacylglycerol hydrolase: Role in intracellular lipid metabolism. Cell Mol. Life Sci. 61:1633‐1651.
   Godin, S.J., Scollon, E.J., Hughes, M.F., Potter, P.M., DeVito, M.J., and Ross, M.K. 2006. Species differences in the in vitro metabolism of deltamethrin and esfenvalerate: Differential oxidative and hydrolytic metabolism by humans and rats. Drug Metab. Dispos. 34:1764‐1771.
   Guengerich, F.P. 2001. Analysis and characterization of enzymes and nucleic acids. In Principles and Methods of Toxicology, 4th ed. pp. 1625‐1687. (A.W. Hayes, ed.) Taylor and Francis, Philadelphia.
   Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680‐685.
   Li, B., Sedlacek, M., Manoharan, I., Boopathy, R., Duysen, E.G., Masson, P., and Lockridge, O. 2005. Butyrylcholinesterase, paraoxonase, and albumin esterase, but not carboxylesterase, are present in human plasma. Biochem. Pharmacol. 70:1673‐1684.
   Morgan, E.W., Yan, B., Greenway, D., Petersen, D.R., and Parkinson, A. 1994. Purification and characterization of two rat liver microsomal carboxylesterases (hydrolase A and B). Arch. Biochem. Biophys. 315:495‐512.
   Mori, M., Hosokawa, M., Ogasawara, Y., Tsukada, E., and Chiba, K. 1999. cDNA cloning, characterization and stable expression of novel human brain carboxylesterase. FEBS Lett. 458:17‐22.
   Morton, C.L. and Potter, P.M. 2000. Comparison of Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Spodoptera frugiperda, and COS7 cells for recombinant gene expression. Application to a rabbit liver carboxylesterase. Mol. Biotechnol. 16:193‐202.
   Pindel, E.V., Kedishvili, N.Y., Abraham, T.L., Brzezinski, M.R., Zhang, J., Dean, R.A., and Bosron, W.F. 1997. Purification and cloning of a broad substrate specificity human liver carboxylesterase that catalyzes the hydrolysis of cocaine and heroin. J. Biol. Chem. 272:14769‐14775.
   Potter, P.M. and Wadkins, R.M. 2006. Carboxylesterases: Detoxifying enzymes and targets for drug therapy. Curr. Med. Chem. 13:1045‐1054.
   Potter, P.M, Pawlik, C.A., Morton, C.L., Naeve, C.W., and Danks, M.K. 1998. Isolation and partial characterization of a cDNA encoding a rabbit liver carboxylesterase that activates the prodrug irinotecan (CPT‐11). Cancer Res. 58:2646‐2651.
   Robbi, M. and Beaufay, H. 1991. The COOH terminus of several liver carboxylesterases targets these enzymes to the lumen of the endoplasmic reticulum. J. Biol. Chem. 266:20498‐20503.
   Ross, M.K., Borazjani, A., Edwards, C.C., and Potter, P.M. 2006. Hydrolytic metabolism of pyrethroids by human and other mammalian carboxylesterases. Biochem. Pharmacol. 71:657‐669.
   Satoh, T. and Hosokawa, M. 1998. The mammalian carboxylesterases: From molecules to functions. Annu. Rev. Pharmacol. Toxicol. 38:257‐288.
   Satoh, T., Taylor, P., Bosron, W.F., Sanghani, S.P., Hosokawa, M., and La Du, B.N. 2002. Current progress on esterases: From molecular structure to function. Drug Metab. Dispos. 30:488‐493.
   Wu, M.H., Chen, P., Remo, B.F., Cook, E.H. Jr, Das, S., and Dolan, M.E. 2003. Characterization of multiple promoters in the human carboxylesterase 2 gene. Pharmacogenetics 13:425‐435.
   Zhao, B., Song, J., St. Clair, R., and Ghosh, S. 2007. Stable over‐expression of human macrophage cholesteryl ester hydrolase (CEH) results in enhanced free cholesterol efflux from human THP1‐macrophages. Am. J. Physiol. Cell Physiol. 292:C405‐C412.
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