Methods for Measuring Cysteine S‐Conjugate β‐Lyase Activity

Lawrence H. Lash1

1 Wayne State University School of Medicine, Detroit, Michigan
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 6.13
DOI:  10.1002/0471140856.tx0613s34
Online Posting Date:  November, 2007
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Abstract

The cysteine conjugate β‐lyase represents activities in cytoplasm and mitochondria catalyzed by at least eleven pyridoxal 5′‐phosphate (PLP)‐dependent enzymes in various tissues. These enzymes mediate bioactivation of cysteine S‐conjugates of several haloalkanes and haloalkenes. The reaction occurs through either a direct β‐elimination or a transamination followed by a retro‐Michael rearrangement, resulting in the cleavage of a C–S bond. The resultant product is a reactive thiolate that rearranges to form thioacylating species. This unit presents several protocols for the assay of β‐lyase activity and includes measurements of product formation and substrate loss as well as fluorescent activity stains. Support protocols describe the synthesis and high‐performance liquid chromatography analysis of selected cysteine S‐conjugates. Because of the diversity of enzymes that can catalyze a β‐lyase reaction, each of the assays presented here may indicate only a portion of the potential β‐lyase activity in a given biological preparation. Curr. Protoc. Toxicol. 34:6.13.1‐6.13.22. © 2007 by John Wiley & Sons, Inc.

Keywords: glutathione conjugation pathway; cysteine S‐conjugate β‐lyase; β‐elimination; transamination; aminotransferases; glutamine transaminase K; kynureninase; reactive thiolate; pyruvate; spectrophotometric assays; HPLC assays

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Assay of Cysteine S–Conjugate β‐Lyase with S‐(2‐Benzothiazolyl)‐L‐Cysteine
  • Basic Protocol 2: Assay of Glutamine Transaminase K Activity
  • Alternate Protocol 1: Glutamine Transaminase K and Cysteine S‐Conjugate β‐Lyase Activity Stains
  • Basic Protocol 3: High‐Performance Liquid Chromatography (HPLC) Assay of Pyruvic Acid Formation
  • Alternate Protocol 2: Assay of Pyruvic Acid Formation by Spectrophotometric Method
  • Support Protocol 1: Synthesis of S‐(2‐Benzothiazolyl)‐L‐Cysteine (BTC)
  • Support Protocol 2: Synthesis of S‐(1,2‐Dichlorovinyl)‐L‐Cysteine (DCVC)
  • Support Protocol 3: Synthesis of S‐(1,2,2‐Trichlorovinyl)‐L‐Cysteine (TCVC)
  • Support Protocol 4: High‐Performance Liquid Chromatography Analysis of S‐(1,2‐Dichlorovinyl)‐L‐Cysteine (DCVC)
  • Support Protocol 5: Nondenaturing Polyacrylamide Gel Electrophoresis (ND‐PAGE)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Assay of Cysteine S–Conjugate β‐Lyase with S‐(2‐Benzothiazolyl)‐L‐Cysteine

  Materials
  • 4 mM S‐(2‐benzothiazolyl)‐L‐cysteine (BTC) solution (see recipe)
  • 0.1 M potassium borate buffer, pH 8.6 (see recipe)
  • 0.2 ml enzyme sample (e.g., subcellular fraction, cellular homogenate, purified enzyme preparation)/time point tested
  • 10% (w/v) trichloroacetic acid
  • 25‐ml polypropylene Erlenmeyer flasks (e.g., Nalgene)
  • Shaking metabolic incubator (e.g., Dubnoff), 37°C
  • Timer
  • 11‐ml (16 × 125–mm) polycarbonate centrifuge tubes (e.g., Pyrex)
  • Table‐top clinical centrifuge (e.g., Dynac)
  • 1.5‐ml or 4.5‐ml polystyrene cuvettes, 10‐cm light path (e.g., Fisherbrand)
  • UV/visible variable wavelength spectrophotometer

Basic Protocol 2: Assay of Glutamine Transaminase K Activity

  Materials
  • 100 mM L‐phenylalanine (Phe)
  • 100 mM α‐keto‐γ‐methiolbutyrate (KMB), sodium salt
  • 1 M 2‐amino‐2‐methyl‐1,3‐propanediol HCl, pH 9.0 (ammediol‐HCl; filtered through activated charcoal)
  • 0.02 ml enzyme sample (e.g., purified enzyme, cell, tissue, or subcellular fraction homogenate) per time point tested
  • 3.3 M NaOH
  • 25‐ml polypropylene Erlenmeyer flasks (e.g., Nalgene)
  • Shaking metabolic incubator (e.g., Dubnoff), 37°C
  • 11‐ml (16 × 125–mm) polycarbonate centrifuge tubes (e.g., Pyrex)
  • 1.5‐ml or 4.5‐ml polystyrene cuvettes, 10 cm light path (e.g., Fisherbrand)
  • UV/visible, variable wavelength spectrophotometer

Alternate Protocol 1: Glutamine Transaminase K and Cysteine S‐Conjugate β‐Lyase Activity Stains

  Materials
  • Enzyme samples (e.g., tissue homogenates containing 50 to 100 µg/ml protein)
  • 100 mM potassium phosphate buffer, pH 7.2 ( appendix 2A)
  • 20 mM L‐phenlyalanine (Phe; for GTK stain) or 2 mM S‐(1,2‐dichlorovinyl)‐L‐cysteine (DCVC; see protocol 7; for β‐lyase stain)
  • 5 mM α‐keto‐γ‐methiolbutyrate (KMB)
  • 32 mM NAD+ (for GTK stain)
  • 0.1 mM phenazine methosulfate (PMS)
  • 1 mM nitroblue tetrazolium (NBT)
  • 12 U/ml GDH (for GTK stain; e.g., from bovine liver)
  • Gel fixation solution: 40:7:53 (v/v/v) methanol/acetic acid/water
  • 0.1% (w/v) Coomassie blue staining solution in gel fixation solution
  • Light box
  • Camera or digital imaging equipment
  • Additional reagents and equipment for performing nondenaturing polyacrylamide electrophoresis ( protocol 10)

Basic Protocol 3: High‐Performance Liquid Chromatography (HPLC) Assay of Pyruvic Acid Formation

  Materials
  • β‐lyase enzyme source (e.g., tissue homogenate or subcellular fraction)
  • 50 mM Tris⋅Cl buffer, pH 8.6 (see appendix 2A)
  • Substrate solution: 0.1 to 10 mM cysteine conjugate (e.g., Support Protocols protocol 61, protocol 72, and protocol 83) in 50 mM Tris⋅Cl buffer, pH 8.6 (see appendix 2A)
  • 12 mM o‐phenylenediamine (OPD) in 3 M HCl
  • HPLC eluent: 45:1:54 (v/v/v) methanol/acetic acid/water (use HPLC‐grade reagents and nanopure water)
  • 6.25 µm to 1 mM pyruvate
  • 2‐ml glass incubation vials with rubber caps
  • 25‐G needle
  • 60°C oven
  • Tabletop centrifuge (e.g., Dynac)
  • 100 × 3–mm C18 HPLC column: 5‐µm particle size
  • HPLC system with fluorescence spectrometer

Alternate Protocol 2: Assay of Pyruvic Acid Formation by Spectrophotometric Method

  Materials
  • β‐lyase enzyme source (e.g., isolated cells, purified enzyme, subcellular fraction)
  • 4× NADH solution: 0.4 mM NADH in 50 mM potassium phosphate, pH 7.0
  • 4× substrate solution: 1 to 20 mM cysteine conjugate (e.g., Support Protocols protocol 61, protocol 72, and protocol 83) in 0.4 mM α‐keto‐γ‐methiolbutyrate (KMB)/50 mM potassium phosphate buffer, pH 7.0 (see appendix 2A)
  • 10% (v/v) Triton X‐100 (optional)
  • Lactate dehydrogenase (LDH) solution: dilute stock to 10 U/ml in 50 mM potassium phosphate buffer, pH 7.0 (see appendix 2A)
  • 2× sodium pyruvate standard solution: 0.2 mM sodium pyruvate in 50 mM potassium phosphate buffer, pH 7.0 (see appendix 2A)
  • 1.5‐ml polystyrene cuvettes, 10‐cm light path (e.g., Fisherbrand)
  • UV/visible variable wavelength spectrophotometer with temperature control

Support Protocol 1: Synthesis of S‐(2‐Benzothiazolyl)‐L‐Cysteine (BTC)

  Materials
  • Dry ice
  • Acetone, reagent grade
  • 2‐chlorobenzothiazole (molecular weight = 169.6; boiling point = 141°C; melting point = 21 to 23°C; density = 1.303 g/ml)
  • L‐cysteine, free base
  • Metallic sodium pellets
  • Ether, reagent grade
  • Glacial acetic acid
  • Absolute ethanol
  • Concentrated NH 4OH
  • Thin layer chromatography (TLC) effluent: n‐butanol/acetic acid/water (25:10:4 v/v/v)
  • 0.2% (w/v) ninhydrin in ethanol
  • Synthesis apparatus (see Fig. ) including:
    • Ammonia gas tank (small)
    • Variable voltage, stirring motor
    • Dewar condenser
    • Rubber bowl (to serve as dry ice/acetone bath)
  • 250‐ml round‐bottom, three‐opening flask
  • Decolorizing charcoal
  • Whatman No. 1 filter paper
  • pH paper
  • Vacuum source
  • 5 × 20–cm thin layer chromatography (TLC) plate
NOTE: All steps should be performed in a properly vented hood to prevent exposure to ammonia gas.

Support Protocol 2: Synthesis of S‐(1,2‐Dichlorovinyl)‐L‐Cysteine (DCVC)

  • Acetone, reagent grade
  • Trichloroethylene
  • L‐cysteine, free base

Support Protocol 3: Synthesis of S‐(1,2,2‐Trichlorovinyl)‐L‐Cysteine (TCVC)

  • Perchloroethylene
  • 80% (v/v) acetonitrile in water
  • Silica gel column
For this protocol follow protocol 7 with the following changes:

Support Protocol 4: High‐Performance Liquid Chromatography Analysis of S‐(1,2‐Dichlorovinyl)‐L‐Cysteine (DCVC)

  Materials
  • Sample of interest (e.g., tissue specimen or incubation mixture) for quantifying DCVC content
  • 1.5 µM γ‐glutamyl‐L‐glutamate in 0.3% (v/v) perchloric acid (internal standard)
  • 15 mM bathophenanthroline disulfonate (BPDS) in metal‐free water (antioxidant)
  • Perchloric acid, 70% (v/v)
  • 100 mM iodoacetic acid (IAA): prepare fresh
  • 2 M KOH/2.4 M KHCO 3 solution
  • 1% (v/v) 1‐fluoro‐2,4‐dinitrobenzene (FDNB) in ethanol: prepare fresh
  • Mobile phase A: 80% (v/v) aqueous methanol
  • Mobile phase B: 0.5 M sodium acetate in 64% (v/v) aqueous methanol; prepare by adding 200 ml sodium acetate solution (see recipe) to 800 ml HPLC mobile phase A
  • 1.5‐ml polypropylene microcentrifuge tubes
  • Microcentrifuge
  • 16 × 125–mm (11 ml) glass test tubes
  • Aluminum foil
  • Tabletop clinical centrifuge (e.g., Dynac)
  • Reversed‐phase, ion‐exchange HPLC column (e.g., 4.6 mm × 25 cm column or 8 mm × 10 cm cartridges; Waters): µ‐Bondapak amine 10‐µm pore‐size
  • Gradient HPLC system with UV/visible detector

Support Protocol 5: Nondenaturing Polyacrylamide Gel Electrophoresis (ND‐PAGE)

  Materials
  • Separating gel buffer: 350 mM Tris⋅Cl, pH 7.35 (see appendix 2A)
  • Enzyme samples (e.g., tissue homogenates containing 50 to 100 µg/ml protein)
  • Sample buffer: 50 mM Tris⋅Cl/200 mM glycine, pH 8.3 (see appendix 2A)/20% (v/v) glycerol/0.001% (w/v) bromphenol blue
  • Molecular weight marker proteins:
    • thyroglobulin dimer (M r = 1,340 kDa)
    • thyroglobulin monomer (M r = 670 kDa)
    • ferritin (M r = 440 kDa)
    • catalase (M r = 232 kDa)
    • lactate dehydrogenase (M r = 140 kDa)
    • albumin (M r = 67 kDa)
  • Electrophoresis buffer: 25 mM Tris⋅Cl, pH 8.3 (see appendix 2A)/100 mM glycine
  • Gel staining solution: 0.1% (w/v) Coomassie blue in methanol/acetic acid/water (40:7:53 v/v/v)
  • Gel destaining solution: methanol/acetic acid/water (5:7.5:87.5 v/v/v)
  • 16 × 15–cm slab gel electrophoresis apparatus
  • Additional reagents and equipment for preparing a polyacrylamide, linear gradient, slab gel ( appendix 3F)
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Figures

Videos

Literature Cited

   Abraham, D.G. and Cooper, A.J.L. 1991. Glutamine transaminase K and cysteine S‐conjugate β‐lyase activity stains. Anal. Biochem. 197:421‐427.
   Abraham, D.G., Patel, P.P., and Cooper, A.J.L. 1995a. Isolation from rat kidney of a cytosolic high molecular weight cysteine S‐conjugate β‐lyase with activity toward leukotriene E4. J. Biol. Chem. 270:180‐188.
   Abraham, D.G., Thomas, R.J., and Cooper, A.J.L. 1995b. Glutamine transaminase K is not a major cysteine S‐conjugate β‐lyase of rat kidney mitochondria: Evidence that a high‐molecular weight enzyme fulfills this role. Mol. Pharmacol. 48:855‐860.
   Anderson, P.M. and Schultze, M.O. 1965. Cleavage of S‐(1,2‐dichlorovinyl)‐L‐cysteine by an enzyme of bovine origin. Arch. Biochem. Biophys. 111:593‐602.
   Bhattacharya, R.K. and Schultze, M.O. 1967. Enzymes from bovine and turkey kidneys which cleave S‐(1,2‐dichlorovinyl)‐L‐cysteine. Comp. Biochem. Physiol. 22:723‐735.
   Cooper, A.J.L. 1978. Purification of soluble and mitochondrial glutamine transaminase K from rat kidney. Anal. Biochem. 89:451‐460.
   Cooper, A.J.L. and Meister, A. 1985. Glutamine transaminase K from rat kidney. Meth. Enzymol. 113:344‐349.
   Cooper, A.J.L. and Pinto, J.T. 2006. Cysteine S‐conjugate β‐lyases. Amino Acids 30:1‐15.
   Cooper, A.J.L., Wang, J., Gartner, C.A., and Bruschi, S.A. 2001. Copurification of mitochondrial HSP70 and mature protein disulfide isomerase with a functional rat kidney high‐Mr cysteine S–conjugate β‐lyase. Biochem. Pharmacol. 62:1345‐1353.
   Cooper, A.J.L., Bruschi, S.A., Iriarte, A., and Martinez‐Carrion, M. 2002. Mitochondrial aspartate aminotransferase catalyses cysteine S‐conjugate β‐lyase reactions. Biochem. J. 368:253‐261.
   Cooper, A.J.L., Bruschi, S.A., Conway, M., and Hutson, S.M. 2003. Human mitochondrial and cytosolic branched‐chain aminotransferases are cysteine S‐conjugate β‐lyases, but turnover leads to inactivation. Biochem. Pharmacol. 65:181‐192.
   Cummings, B.S. and Lash, L.H. 2000. Metabolism and toxicity of trichloroethylene and S‐(1,2‐dichlorovinyl)‐L‐cysteine in freshly isolated human proximal tubular cells. Toxicol. Sci. 53:458‐466.
   Davis, B.J. 1964. Disc electrophoresis. II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121:404‐427.
   Dekant, W., Berthold, K., Vamvakas, S., Henschler, D., and Anders, M.W. 1988. Thioacylating intermediates as metabolites of S‐(1,2‐dichlorovinyl)‐L‐cysteine and S‐(1,2,2‐trichlorovinyl)‐L‐cysteine formed by cysteine conjugate β‐lyase. Chem. Res. Toxicol. 1:175‐178.
   Dohn, D.R. and Anders, M.W. 1982. Assay of cysteine conjugate β‐lyase activity with S‐(2‐benzothiazolyl)cysteine as the substrate. Anal. Biochem. 120:379‐386.
   Elfarra, A.A. and Hwang, I.Y. 1990. In vivo metabolites of S‐(2‐benzothiazolyl)‐L‐cysteine as markers of in vivo cysteine conjugate β‐lyase and thiol glucuronosyl transferase activities. Drug Metab. Dispos. 18:917‐922.
   Elfarra, A.A., Lash, L.H., and Anders, M.W. 1987. Alpha‐keto acids stimulate rat renal cysteine conjugate β‐lyase activity and potentiate the cytotoxicity of S‐(1,2‐dichlorovinyl)‐L‐cysteine. Mol. Pharmacol. 31:208‐212.
   Fariss, M.W. and Reed, D.J. 1987. High‐performance liquid chromatography of thiols and disulfides: Dinitrophenyl derivatives. Meth. Enzymol. 143:101‐109.
   Hayden, P., Schaeffer, V.H., Larsen, G., and Stevens, J.L. 1987. Cysteine S‐conjugates. Meth. Enzymol. 143:228‐234.
   Hwang, I.Y. and Elfarra, A.A. 1989. Cysteine S‐conjugates may act as kidney‐selective prodrugs: Formation of 6‐mercaptopurine by the renal metabolism of S‐(6‐purinyl)‐L‐cysteine. J. Pharmacol. Exp. Ther. 251:448‐454.
   Hwang, I.Y. and Elfarra, A.A. 1991. Kidney‐selective prodrugs of 6‐mercaptopurine: Biochemical basis of kidney selectivity of S‐(6‐purinyl)‐L‐cysteine and metabolism of new analogs in rats. J. Pharmacol. Exp. Ther. 258:171‐177.
   Jones, T.W., Qin, C., Schaeffer, V.H., and Stevens, J.L. 1988. Immunohistochemical localization of glutamine transaminase K, a rat kidney cysteine conjugate β‐lyase, and the relationship to the segment specificity of cysteine conjugate nephrotoxicity. Mol. Pharmacol. 34:621‐627.
   Krause, R.J., Lash, L.H., and Elfarra, A.A. 2003. Human kidney flavin‐containing monooxygenases and their potential roles in cysteine S–conjugate metabolism and nephrotoxicity. J. Pharmacol. Exp. Ther. 304:185‐191.
   Lash, L.H. and Anders, M.W. 1986. Cytotoxicity of S‐(1,2‐dichlorovinyl)glutathione and S‐(1,2‐dichlorovinyl)‐L‐cysteine in isolated rat kidney cells. J. Biol. Chem. 261:13076‐13081.
   Lash, L.H. and Anders, M.W. 1989. Uptake of nephrotoxic S‐conjugates by isolated rat renal proximal tubular cells. J. Pharmacol. Exp. Ther. 248:531‐537.
   Lash, L.H. and Jones, D.P. 1985. Uptake of the glutathione conjugate S‐(1,2‐dichlorovinyl)glutathione by renal basal‐lateral membrane vesicles and isolated kidney cells. Mol. Pharmacol. 28:278‐282.
   Lash, L.H., Elfarra, A.A., and Anders, M.W. 1986. Renal cysteine conjugate β‐lyase: Bioactivation of nephrotoxic cysteine S‐conjugates in mitochondrial outer membrane. J. Biol. Chem. 261:5930‐5935.
   Lash, L.H., Nelson, R.M., Van Dyke, R.A., and Anders, M.W. 1990. Purification and characterization of human kidney cytosolic cysteine conjugate β‐lyase activity. Drug Metab. Dispos. 18:50‐54.
   Lash, LH, Sausen, P.J., Duescher, R.J., Cooley, A.J., and Elfarra, A.A. 1994. Roles of cysteine conjugate β‐lyase and S‐oxidase in nephrotoxicity: Studies with S‐(1,2‐dichlorovinyl)‐L‐cysteine and S‐(1,2‐dichlorovinyl)‐L‐cysteine sulfoxide. J. Pharmacol. Exp. Ther. 269:374‐383.
   Lash, L.H., Shivnani, A., Mai, J., Chinnaiyan, P., Krause, R.J., and Elfarra, A.A. 1997. Renal cellular transport, metabolism and cytotoxicity of S‐(6‐purinyl)glutathione, a prodrug of 6‐mercaptopurine, and analogues. Biochem. Pharmacol. 54:1341‐1349.
   Lash, L.H., Fisher, J.W., Lipscomb, J.C., and Parker, J.C. 2000a. Metabolism of trichloroethylene. Environ. Health Perspect. 108:177‐200.
   Lash, L.H., Parker, J.C., and Scott, C.S. 2000b. Modes of action of trichloroethylene for kidney tumorigenesis. Environ. Health Perspect. 108:225‐240.
   Lash, L.H., Hueni, S.E., and Putt, D.A. 2001. Apoptosis, necrosis and cell proliferation induced by S‐(1,2‐dichlorovinyl)‐L‐cysteine in primary cultures of human proximal tubular cells. Toxicol. Appl. Pharmacol. 177:1‐16.
   Lash, L.H., Putt, D.A., Hueni, S.E., Krause, R.J., and Elfarra, A.A. 2003. Roles of necrosis, apoptosis, and mitochondrial dysfunction in S‐(1,2‐dichlorovinyl)‐L‐cysteine sulfoxide‐induced cytotoxicity in primary cultures of human renal proximal tubular cells. J. Pharmacol. Exp. Ther. 305:1163‐1172.
   Lash, L.H., Putt, D.A., Hueni, S.E., and Horwitz, B.P. 2005. Molecular markers of trichloroethylene‐induced toxicity in human kidney cells. Toxicol. Appl. Pharmacol. 206:157‐168.
   MacFarlane, M., Schofield, M., Parker, N., Roelandt, L., David, M., Lock, E.A., King, L.J., Goldfarb, P.S., and Gibson, G.G. 1993. Dose‐dependent induction or depression of cysteine conjugate β‐lyase in rat kidney by N‐acetyl‐S‐(1,2,3,4,4‐pentachloro‐1,3‐butadienyl)‐L‐cysteine. Toxicology 77:133‐144.
   McKinney, L.L., Picken, J.C. Jr., Weakley, F.B., Eldridge, A.C., Campbell, R.E., Cowan, J.C., and Biester, H.E. 1959a. Possible toxic factor of trichloroethylene‐extracted soybean oil meal. J. Am. Chem. Soc. 81:909‐915.
   McKinney, L.L., Eldridge, A.C., and Cowan, J.C. 1959b. Cysteine thioethers from chloroethylenes. J. Am. Chem. Soc. 81:1423‐1427.
   National Toxicology Program (NTP). 2002. Report on carcinogens, 10th ed. U.S. Department of Health and Human Services, Public Health Service, National Toxicology Program, Research Triangle Park, N.C.
   Perry, S.J., Schofield, M.A., Macfarlane, M., Lock, E.A., King, L.J., Gibson, G.G., and Goldfarb, P.S. 1993. Isolation and expression of a cDNA coding for rat kidney cysteine conjugate β‐lyase. Mol. Pharmacol. 43:660‐665.
   Perry, S.J., Harries, H., Scholfield, C., Lock, T., King, L., Gibson, G., and Goldfarb, P. 1995. Molecular cloning and expression of a cDNA for human kidney cysteine conjugate β‐lyase. FEBS Lett. 360:277‐280.
   Reed, D.J., Babson, J.R., Beatty, P.W., Brodie, A.E., Ellis, W.W., and Potter, D.W. 1980. High‐performance liquid chromatography analysis of nanomole levels of glutathione, glutathione disulfide, and related thiols and disulfides. Anal. Biochem. 106:55‐62.
   Ripp, S.L., Overby, L.H., Philpot, R.M., and Elfarra, A.A. 1997. Oxidation of cysteine S‐conjugates by rabbit liver microsomes and cDNA‐expressed flavin‐containing monooxygenases: Studies with S‐(1,2‐dichlorovinyl)‐L‐cysteine, S‐(1,2,2‐trichlorovinyl)‐L‐cysteine, S‐allyl‐L‐cysteine, and S‐benzyl‐L‐cysteine. Mol. Pharmacol. 51:507‐515.
   Sausen, P.J. and Elfarra, A.A. 1990. Cysteine conjugate S‐oxidase: Characterization of a novel enzymatic activity in rat hepatic and renal microsomes. J. Biol. Chem. 265:6139‐6145.
   Sausen, P.J., Duescher, R.J., and Elfarra, A.A. 1993. Further characterization and purification of the flavin‐dependent S‐benzyl‐L‐cysteine S‐oxidase activities of rat liver and kidney microsomes. Mol. Pharmacol. 43:388‐396.
   Stevens, J.L. 1985. Isolation and characterization of a rat liver enzyme with both cysteine conjugate β‐lyase and kynureninase activity. J. Biol. Chem. 260:7945‐7950.
   Stevens, J.L. and Jakoby, W.B. 1983. Cysteine conjugate β‐lyase. Mol. Pharmacol. 23:761‐765.
   Stevens, J.L. and Jakoby, W.B. 1985. Cysteine conjugate β‐lyase. Meth. Enzymol. 113:510‐515.
   Stevens, J.L., Robbins, J.D., and Byrd, R.A. 1986. A purified cysteine conjugate β‐lyase from rat kidney cytosol: Requirement for an α‐keto acid or an amino acid oxidase for activity and identity with soluble glutamine transaminase K. J. Biol. Chem. 261:15529‐15537.
   Stijntjes, G.J., te Koppele, J.M., and Vermeulen, N.P.E. 1992. High‐performance liquid chromatography‐fluorescence assay of pyruvic acid to determine cysteine conjugate β‐lyase activity: Application to S‐(1,2‐dichlorovinyl)‐L‐cysteine and S‐2‐benzothiazolyl‐L‐cysteine. Anal. Biochem. 206:334‐343.
   Tateishi, M., Suzuki, S., and Shimuzu, H. 1978. Cysteine conjugate β‐lyase in rat liver: A novel enzyme catalyzing formation of thiol‐containing metabolites of drugs. J. Biol. Chem. 253:8854‐8859.
Key References
   Cooper and Pinto, 2006. See above.
  This is the most recent review that has focused on the various and diverse enzymes that constitute what are called the cysteine S‐conjugate β‐lyases.
   Lash et al., 2000a. See above.
  These two references present a comprehensive summary of metabolism and mode of action for renal toxicity of trichloroethylene, an important environmental contaminant; the cysteine conjugate DCVC is metabolized, at least in part, by the β‐lyase to a nephrotoxic species.
   Lash et al., 2000b. See above.
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