Measurement of Cysteine Dioxygenase Activity and Protein Abundance

Martha H. Stipanuk1, John E. Dominy Jr.1, Iori Ueki1, Lawrence L. Hirschberger1

1 Division of Nutritional Sciences, Cornell University, Ithaca, New York
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 6.15
DOI:  10.1002/0471140856.tx0615s38
Online Posting Date:  November, 2008
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Cysteine dioxygenase is an iron (Fe2+)–dependent thiol dioxygenase that uses molecular oxygen to oxidize the sulfhydryl group of cysteine to generate 3‐sulfinoalanine (commonly called cysteinesulfinic acid). Cysteine dioxygenase activity is routinely assayed by measuring cysteinesulfinate formation from substrate L‐cysteine at pH 6.1 in the presence of ferrous ions to saturate the enzyme with metal cofactor, a copper chelator to diminish substrate oxidation, and hydroxylamine to inhibit pyridoxal 5′‐phosphate‐dependent degradation of product. The amount of cysteine dioxygenase may be measured by immunoblotting. Upon SDS‐PAGE, cysteine dioxygenase can be separated into two major bands, with the upper band representing the 23‐kDa protein and the lower band representing the mature enzyme that has undergone formation of an internal thioether cross‐link in the active site. Formation of this cross‐link is dependent upon the catalytic turnover of substrate and produces an enzyme with a higher catalytic efficiency and catalytic half‐life. Curr. Protoc. Toxicol. 38:6.15.1‐6.15.25. © 2008 by John Wiley & Sons, Inc.

Keywords: cysteine dioxygenase; cysteine; cysteinesulfinic acid; taurine; sulfate

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Standard Assay for Cysteine Dioxygenase Activity in Tissue Samples or Cell Extracts
  • Alternate Protocol 1: Simplified Assay for Measurement of Activity of Purified Cysteine Dioxygenase
  • Alternate Protocol 2: Radiolabeled Assay for Cysteine Dioxygenase Activity
  • Basic Protocol 2: Western Blotting to Measure CDO Protein Levels
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Standard Assay for Cysteine Dioxygenase Activity in Tissue Samples or Cell Extracts

  Materials
  • Sample: liver tissue, isolated hepatocytes or other cells in suspension, or primary or other attached cell cultures
  • 50 mM MES buffer, pH 6.1 (see recipe)
  • 200 mM MES buffer, pH 6.1 (see recipe)
  • Fe(II)/NAD/hydroxylamine premix (see recipe)
  • 20 mM cysteine/0.25 mM BCS solution (see recipe)
  • 5% sulfosalicylic acid (SSA; see recipe)
  • Dowex 50 WX8 resin suspension (see recipe)
  • 0.10 mM L‐asparagine (see recipe)
  • 0.2 M borate buffer, pH 10.4 (see recipe)
  • OPA derivitization mixture (see recipe)
  • 1.0 mM potassium phosphate, pH 7.0 (see recipe)
  • Buffer A (see recipe)
  • Buffer B (see recipe)
  • L‐cysteinesulfinic acid (CSA) standard curve solutions (see recipe)
  • L‐cysteic acid (CA) standard curve solutions (see recipe)
  • Refrigerated centrifuge
  • Tissue homogenizer: either rotor/stator or Dounce homogenizer
  • Ultrasonic processor/sonicator with microtip (e.g., Vibra Cell Ultrasonic Processor, Sonics & Materials, Inc.; http://www.sonicsandmaterials.com)
  • Eppendorf Thermomixer (Brinkmann Instruments)
  • HPLC system with automatic sample injector (e.g., WISP model 717 plus, Waters Corporation) and fluorescence detector (e.g., Model 2475, Waters Corporation)
  • Nova‐Pak C 18, 4.6 × 150–mm column for HPLC (4 µm spherical particles; Waters Corporation)
  • C 18 guard column cartridge for HPLC (5 µm spherical particles; Alltech)
  • PC running Empower 2 software (Waters Corporation)
  • Additional reagents and equipment for protein assay ( appendix 3I)

Alternate Protocol 1: Simplified Assay for Measurement of Activity of Purified Cysteine Dioxygenase

  • 2.5 mM ferrous sulfate solution (see recipe)
  • Source of purified CDO protein
  • 12 mM cysteine/0.25 mM BCS solution (see recipe)

Alternate Protocol 2: Radiolabeled Assay for Cysteine Dioxygenase Activity

  Materials
  • Tissue or cells of interest
  • TNES lysis buffer with protease inhibitors (see recipe)
  • 6× loading buffer (see recipe)
  • Bincinchoninic acid protein assay kit (Pierce; also see appendix 3I)
  • Protein molecular weight marker (see appendix 3F)
  • SDS‐polyacrylamide gel: 12% or 15% (w/v) acrylamide (see appendix 3F and annotation to step 5, below)
  • 1× electrode buffer (see recipe)
  • Transfer buffer (see recipe)
  • Blocking buffer (see recipe)
  • Primary antibody (anti‐CDO): Anti–rat/mouse CDO from the authors' laboratory is available from Abcam as the IgG fraction of the rabbit anti‐CDO immune serum
  • Control protein antibody, e.g., anti‐actin
  • 1× TBST (see recipe)
  • Secondary antibody: horseradish peroxidase–conjugated goat anti‐rabbit secondary antibody (e.g., Pierce, cat. no. 1858415)
  • Chemiluminescent substrate (e.g., Supersignal West Pico or Supersignal West Dura from Thermo Scientific)
  • Restore Western Blot Stripping Buffer (Pierce)
  • Tissue homogenizer: either rotor/stator or Dounce homogenizer
  • Refrigerated centrifuge
  • 95° to 100°C water bath or heating block
  • Electrophoresis apparatus and power source ( appendix 3F)
  • Polyvinylidine difluoride (PVDF) membranes: e.g., 0.45‐µm Immobilon‐P membranes (Millipore)
  • Electroblotting apparatus (see Gallagher et al., )
  • Adjustable speed rocking platform
  • X‐ray film
  • Desktop scanner and software (e.g., NIH Image 1.63 software)
  • Additional reagents and equipment for protein assay ( appendix 3I), SDS‐PAGE ( appendix 3F), and immunoblotting (Gallagher et al., )
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Bagley, P.J. and Stipanuk, M.H. 1994. The activities of hepatic cysteine dioxygenase and cysteinesulfinate decarboxylase are regulated in a reciprocal manner in response to dietary casein level in rats. J. Nutr. 124:2410‐2421.
   Bagley, P.J., Hirschberger, L.L., and Stipanuk, M.H. 1995. Evaluation and modification of an assay procedure for cysteine dioxygenase activity: High‐performance liquid chromatography method for measurement of cysteine sulfinate and demonstration of physiological relevance of cysteine dioxygenase activity in cysteine catabolism. Anal. Biochem. 227:40‐48.
   Bella, D.L., Kwon, Y.H., and Stipanuk, M.H. 1996. Variations in dietary protein but not in dietary fat plus cellulose or carbohydrate levels affect cysteine metabolism in rat isolated hepatocytes. J. Nutr. 126:2179‐2187.
   Bella, D.L., Hirschberger, L.L., Hosokawa, Y., and Stipanuk, M.H. 1999. The mechanisms involved in the regulation of key enzymes of cysteine metabolism in rat liver in vivo. Am. J. Physiol. 276:E326‐E335.
   Coloso, R.M., Drake, M.R., and Stipanuk, M.H. 1990. Effect of bathocuproine disulfonate, a copper chelator, on cyst(e)ine metabolism by freshly isolated rat hepatocytes. Am. J. Physiol. 259:E443‐E450.
   Daniels, K.M. and Stipanuk, M.H. 1982. The effect of dietary cysteine level on cysteine metabolism in rats. J. Nutr. 112:2130‐2141.
   de la Rosa, J. and Stipanuk, M.H. 1985. Evidence for a rate‐limiting role of cysteinesulfinate decarboxylase activity in taurine biosynthesis in vivo. Comp. Biochem. Physiol. B. 81:565‐571.
   Dominy, J.E. Jr., Hirschberger, L.L., Coloso, R.M., and Stipanuk, M.H. 2006a. Regulation of cysteine dioxygenase degradation is mediated by intracellular cysteine levels and the ubiquitin‐26S proteasome system in the living rat. Biochem. J. 394:267‐273.
   Dominy, J.E. Jr., Simmons, C.R., Karplus, P.A., Gehring, A.M., and Stipanuk, M.H. 2006b. Identification and characterization of bacterial cysteine dioxygenases: A new route of cysteine degradation for eubacteria. J. Bacteriol. 188:5561‐5569.
   Dominy, J.E. Jr., Hwang, J., and Stipanuk, M.H. 2007. Overexpression of cysteine dioxygenase reduces intracellular cysteine and glutathione pools in HepG2/C3A cells. Am. J. Physiol. Endocrinol. Metab. 293:E62‐E69.
   Dominy, J.E. Jr., Hwang, J., Guo, S., Hirschberger, L.L., Zhang, S., and Stipanuk, M.H. 2008. Synthesis of amino acid cofactor in cysteine dioxygenase is regulated by substrate and represents a novel post‐translational regulation of activity. J. Biol. Chem. 283:12188‐12201.
   Gallagher, S., Winston, S.E., Fuller, S.A., and Hurrell, J.G.R. 2008. Immunoblotting and immunodetection. Curr. Protoc. Mol. Biol. 83:10.8.1‐10.8.28.
   Hirschberger, L.L., Daval, S., Stover, P.J., and Stipanuk, M.H. 2001. Murine cysteine dioxygenase gene: Structural organization, tissue‐specific expression and promoter identification. Gene 277:153‐161.
   Hosokawa, Y., Matsumoto, A., Oka, J., Itakura, H., and Yamaguchi, K. 1990. Isolation and characterization of a cDNA for rat liver cysteine dioxygenase. Biochem. Biophys. Res. Commun. 168:473‐478.
   Kuo, S.M. and Stipanuk, M.H. 1984. Changes in cysteine dioxygenase and cysteinesulfinate decarboxylase activities and taurine levels in tissues of pregnant or lactating rat dams and their fetuses or pups. Biol. Neonate. 46:237‐248.
   Kwon, Y.H. and Stipanuk, M.H. 2001. Cysteine regulates cysteine dioxygenase and γ‐glutamylcysteine synthetase in cultured rat hepatocytes. Am. J. Physiol. 280:E804‐E815.
   Lee, J.‐I., Londono, M.P., Hirschberger, L.L., and Stipanuk, M.H. 2004. Regulation of cysteine dioxygenase and γ‐glutamylcysteine synthetase is associated with hepatic cysteine level. J. Nutr. Biochem. 15:112‐122.
   Lee, J‐I., Kang, J., and Stipanuk, M.H. 2006. Differential regulation of glutamate‐cysteine ligase subunit expression and increased holoenzyme formation in response to cysteine deprivation. Biochem. J. 393:181‐190.
   McCoy, J.G., Bailey, L.J., Bitto, E., Bingman, C.A., Aceti, D.J., Fox, B.G., and Phillips, G.N. Jr. 2006. Structure and mechanism of mouse cysteine dioxygenase. Proc. Natl. Acad. Sci. U.S.A. 103:3084‐3089.
   Ohta, J., Kwon, Y.‐H., and Stipanuk, M.H. 2000. Cysteine metabolism by primary cultures of hepatocytes responds to methionine supplementation of the culture medium. Amino Acids 19:705‐728.
   Parsons, R.B., Barber, P.C., Waring, R.H., Williams, A.C., and Ramsden, D.B. 1998a. Cysteine dioxygenase: Regional expression of activity in rat brain. Neurosci. Lett. 248:101‐104.
   Parsons, R.B., Ramsden, D.B., Waring, R.H., Barber, P.C., and Williams, A.C. 1998b. Hepatic localisation of rat cysteine dioxygenase. J. Hepatol. 29:595‐602.
   Parsons, R.B., Sampson, D., Huggins, C.C., Waring, R.H., Williams, A.C., and Ramsden, D.B. 2001a. Renal localisation of rat cysteine dioxygenase. Nephron 88:340‐346.
   Parsons, R.B., Waring, R.H., Williams, A.C., and Ramsden, D.B. 2001b. Cysteine dioxygenase: Regional localisation of protein and mRNA in rat brain. J. Neurosci. Res. 65:78‐84.
   Qusti, S., Parsons, R.B., Abouglila, K.D., Waring, R.H., Williams, A.C., and Ramsden, D.B. 2000. Development of an in vitro model for cysteine dioxygenase expression in the brain. Cell Biol. Toxicol. 16:243‐255.
   Simmons, C.R., Liu, Q., Huang, Q., Hao, Q., Begley, T.P., Karplus, P.A., and Stipanuk, M.H. 2006a. Crystal structure of mammalian cysteine dioxygenase: A novel mononuclear iron center for cysteine thiol oxidation. J. Biol. Chem. 281:18723‐18733.
   Simmons, C.R., Hirschberger, L.L., Machi, M.S., and Stipanuk, M.H. 2006b. Expression, purification, and kinetic characterization of recombinant rat cysteine dioxygenase, a non‐heme metalloenzyme necessary for regulation of cellular cysteine levels. Protein Expr. Purif. 47:74‐81.
   Stipanuk, M.H. and Beck, P.W. 1982 Characterization of the enzymic capacity for cysteine desulphhydration in liver and kidney of the rat. Biochem. J. 206:267‐277.
   Stipanuk, M.H., Londono, M., Lee, J.‐I., Hu, M., and Yu, A.F. 2002. Enzymes and metabolites of cysteine metabolism in nonhepatic tissues of rats show little response to changes in dietary protein or sulfur amino acid levels. J. Nutr. 132:3369‐3378.
   Stipanuk, M.H., Londono, M., Hirschberger, L.L., Wang, L., and Hickey, C. 2004a. Evidence for expression of a single distinct form of mammalian cysteine dioxygenase. Amino Acids 26:99‐106.
   Stipanuk, M.H., Hirschberger, L.L., Londono, M.P., Cresenzi, C.L., and Yu, A.F. 2004b. The ubiquitin‐proteasome system is responsible for cysteine‐responsive regulation of cysteine dioxygenase concentration in liver. Am. J. Physiol. Endocrinol. Metab. 286:E439‐E448.
   Stipanuk, M.H., Dominy, J.E. Jr., Lee, J.I., and Coloso, R.M. 2006. Mammalian cysteine metabolism: New insights into regulation of cysteine metabolism. J. Nutr. 136:1652S‐1659S.
   Ueki, I. and Stipanuk, M.H. 2007. Enzymes of the taurine biosynthetic pathway are expressed in rat mammary gland. J. Nutr. 137:1887‐1894.
   Yamaguchi, K., Hosokawa, Y., Kohashi, N., Kori, Y., Sakakibara, S., and Ueda, I. 1978. Rat liver cysteine dioxygenase (cysteine oxidase): Further purification, characterization, and analysis of the activation and inactivation. J. Biochem. 83:479‐491.
   Ye, S., Wu, X., Wei, L., Tang, D., Sun, P., Bartlam, M., and Rao, Z. 2007. An insight into the mechanism of human cysteine dioxygenase: Key roles of the thioether‐bonded tyrosine‐cysteine cofactor. J. Biol. Chem. 282:3391‐3402.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library