Measurement of Cysteine S‐Conjugate β‐Lyase Activity

Arthur J.L. Cooper1, Boris F. Krasnikov1, John T. Pinto1, Sam A. Bruschi2

1 New York Medical College, Valhalla, New York, 2 Laural Consulting, Adelaide, Australia
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 4.36
DOI:  10.1002/0471140856.tx0436s44
Online Posting Date:  May, 2010
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Abstract

Cysteine S‐conjugate β‐lyases are pyridoxal 5′‐phosphate (PLP)–containing enzymes that catalyze the conversion of cysteine S‐conjugates [RSCH2CH(NH3+)CO2] and selenium Se‐conjugates [RSeCH2CH(NH3+)CO2] that contain a leaving group in the β position to pyruvate, ammonium and a sulfur‐containing fragment (RSH) or selenium‐containing fragment (RSeH), respectively. In mammals, at least ten PLP enzymes catalyze β‐elimination reactions with such cysteine S‐conjugates. All are enzymes involved in amino acid metabolism that do not normally catalyze a β‐lyase reaction, but catalyze a non‐physiological β‐lyase side‐reaction that depends on the electron‐withdrawing properties of the –SR or –SeR moiety. In the case of cysteine S‐conjugates, if the eliminated RSH is stable, the compound may be S‐thiomethylated and excreted (thiomethyl shunt) or S‐glucuronidated and harmlessly excreted. However, if RSH is chemically reactive, the cysteine S‐conjugate may be toxic as a result of the β‐lyase reaction. The cysteine S‐conjugate β‐lyase pathway is of particular interest to toxicologists because it is involved in the bioactivation (toxification) of halogenated alkenes and certain drugs. This unit provides protocols for the analysis of cysteine S‐conjugate β‐lyase activity. Curr. Protoc. Toxicol. 44:4.36.1‐4.36.18. © 2010 by John Wiley & Sons, Inc.

Keywords: ammonium; cysteine S‐conjugates; cysteine S‐conjugate β‐lyases; S‐(1,2‐dichlorovinyl)‐L‐cysteine; S‐(1,1,2,2‐tetrafluoroethyl)‐L‐cysteine; pyruvate

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Spectrophotometric Measurement of Cysteine S‐Conjugate β‐Lyase Activity Based on Pyruvate as its 2,4‐Dinitrophenylhydrazone
  • Basic Protocol 2: Measurement of Cysteine S‐Conjugate β‐Lyase Activity Using HPLC to Detect Pyruvate as an Azine Derivative
  • Support Protocol 1: Preparation of Cytosol and Mitochondria from Rat Liver
  • Support Protocol 2: Preparation of Cytosol and Mitochondria from Rat Kidney
  • Support Protocol 3: Protein Determination
  • Support Protocol 4: Use of Cystathionine γ‐Lyase as a Cytosolic Marker for Rat Liver Subcellular Fractions
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Spectrophotometric Measurement of Cysteine S‐Conjugate β‐Lyase Activity Based on Pyruvate as its 2,4‐Dinitrophenylhydrazone

  Materials
  • 1 M potassium phosphate buffer, pH 7.2 ( appendix 2A)
  • 1 M Tris⋅Cl buffer, pH 8.5 ( appendix 2A)
  • 100 mM S‐(1,1,2,2‐tetrafluoroethyl)‐L‐cysteine (TFEC; see recipe) in 100 mM potassium phosphate buffer, pH 7.2
  • 50 mM S‐(1,2‐dichlorovinyl)‐L‐cysteine (DCVC; McKinney et al., ) in 100 mM Tris⋅Cl buffer, pH 8.0
  • 1 mM sodium α‐keto‐γ‐methiolbutyrate (Sigma)
  • 1 mM sodium α‐ketoglutarate (Sigma)
  • Enzyme activity source (select one):
    • Tissue homogenate, cytosol, or mitochondrial extract (Support Protocols protocol 31 and protocol 42)
    • Purified rat liver glutamine transaminase K (GTK) or purified recombinant human GTK
    • Purified recombinant rat liver mitochondrial aspartate aminotransferase (mitAspAT)
    • Purified cysteine S‐conjugate β‐lyase
  • 2 mM 2,4‐dinitrophenylhydrazine in 2 M HCl (ICN Biochemicals)
  • 1 M NaOH
  • 0.5‐ml microcentrifuge tubes
  • 37°C heating block
  • 96‐well plates (standard plastic plates) and spectrophotometric plate reader (0.5‐cm path length)
NOTE: GTK and mitAspAT are not available commercially. GTK may be purified from rat kidney by the method of Cooper ( ), Stevens et al. ( ), or Yamauchi et al. ( ). Recombinant human GTK (also known as kynurenine aminotransferase 1 [KAT 1]) may be prepared as described by Han et al. ( ). Recombinant rat liver mitAspAT may be prepared by the method of Mattingly et al. ( ).

Basic Protocol 2: Measurement of Cysteine S‐Conjugate β‐Lyase Activity Using HPLC to Detect Pyruvate as an Azine Derivative

  Materials
  • 1 M potassium phosphate buffer, pH 7.2 ( appendix 2A)
  • 10 mM sodium α‐keto‐γ‐methiolbutyrate
  • Purified enzyme
  • 3 M sodium acetate ( appendix 2A)
  • 0.1% (w/v) 3‐methyl‐2‐benzothiazolinone hydrazone (MBTH) in water (corresponds to 4.6 mM)
  • Stock solution of cysteine S‐conjugate substrate
  • 50% (v/v) acetonitrile in water
  • Mobile phase buffer: 75 mM trisodium citrate/25 mM ammonium acetate/26% (v/v) acetonitrile, pH 7.64
  • 0.5‐ml snap‐top microcentrifuge tubes
  • 37°C heating block
  • HPLC apparatus with electrochemical detection
    • MD‐150 analytical column (3.0 × 150–mm, 3 µm; ESA, Inc.)
    • ESA model 5600 CoulArray module
    • ESA software

Support Protocol 1: Preparation of Cytosol and Mitochondria from Rat Liver

  Materials
  • Young adult rat (4 to 6 months old)
  • Liver mitochondrial isolation buffer (see recipe), ice cold
  • Liver mitochondrial wash buffer (see recipe), ice cold
  • Guillotine
  • 50‐ml glass beakers
  • 8‐cm glass Petri dish
  • Scissors
  • Tweezers
  • 10‐ and 100‐ml Potter homogenizers with Teflon pestles
  • Beckman centrifuge with J20 rotor (or equivalent) and 50‐ml tubes
  • Glass funnel with two layers of cheesecloth
  • Glass rod

Support Protocol 2: Preparation of Cytosol and Mitochondria from Rat Kidney

  Materials
  • Freshly isolated rat kidney
  • Kidney mitochondrial isolation buffer (see recipe), ice cold
  • Kidney mitochondrial wash buffer (see recipe), ice cold
  • Motor‐driven Teflon pestle (Potter‐Elvehjem type)
  • Sorvall centrifuge with HB‐4 high‐speed swinging‐bucket rotor, SS‐34 fixed‐angle rotor, and 50‐ml tubes for each (ThermoScientific) or equivalent
NOTE: All steps are carried out on ice using ice‐cold buffers.

Support Protocol 3: Protein Determination

  Materials
  • Cytosol or mitochondrial suspension (Support Protocols protocol 31 and protocol 42)
  • Total protein Biuret reagent (Sigma; store refrigerated, use at ambient temperature)
  • 100 mg/ml bovine serum albumin (BSA)
  • 96‐well plates and spectrophotometric plate reader

Support Protocol 4: Use of Cystathionine γ‐Lyase as a Cytosolic Marker for Rat Liver Subcellular Fractions

  Materials
  • 1 M potassium phosphate buffer, pH 7.2 ( appendix 2A)
  • 100 mM L‐homoserine (Sigma)
  • Rat liver cytosol and mitochondria ( protocol 3)
  • 2 mM 2,4‐dinitrophenylhydrazine in 2 M HCl (ICN Biochemicals)
  • 1 M NaOH
  • 37°C heating block
  • 96‐well plates and spectrophotometric plate reader
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Figures

Videos

Literature Cited

Literature Cited
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