Complex I Assay in Mitochondrial Preparations from CNS

Latha Diwakar1, Ajit Ray1, Vijayalakshmi Ravindranath1

1 National Brain Research Centre, Nainwal Mode, Manesar,
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 17.10
DOI:  10.1002/0471140856.tx1710s38
Online Posting Date:  November, 2008
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Abstract

Mammalian NADH:ubiquinone oxidoreductase (complex I) is made up of at least 46 subunits and is one of the largest enzyme complexes known. It catalyzes the first step of the respiratory electron transport chain through the oxidation of NADH, providing two electrons for the reduction of ubiquinone to ubiquinol, thus propelling protons across the inner membrane of the mitochondria, which subsequently drive ATP synthesis. Dysfunction of complex I has been implicated in various neurodegenerative disorders, and it is probably the most vulnerable component of the electron transport chain to inhibition by reactive oxygen species. We describe a simple spectrophotometric method for estimating the activity of complex I from mitochondria isolated from regions of the central nervous system of mice. Curr. Protoc. Toxicol. 38:17.10.1‐17.10.7. © 2008 by John Wiley & Sons, Inc.

Keywords: complex I; brain; mitochondria; oxidative stress; electron transport

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Assay of Complex I Activity
  • Support Protocol 1: Preparation of Mitochondrial Suspension from Brain
  • Support Protocol 2: Estimation of Protein Content of Mitochondrial Suspension
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Assay of Complex I Activity

  Materials
  • Mitochondria isolated from mouse brain ( protocol 2), with protein content determined ( protocol 3)
  • Assay buffer for complex I activity (see recipe)
  • 2.5 mM ubiquinone or decylubiquinone in 100% ethanol
  • 500 µM rotenone in 100% ethanol
  • 5 mM β‐NADH (Sigma) in 35 mM potassium phosphate buffer (pH 7.2, appendix 2A), prepared fresh
  • Quartz cuvette(s)
  • UV‐VIS spectrophotometer

Support Protocol 1: Preparation of Mitochondrial Suspension from Brain

  Materials
  • Mouse (3 to 6 months old, 25 to 30 g, male or female)
  • Diethyl ether
  • 0.9% (w/v) sodium chloride (normal saline)
  • Homogenization buffer: 100 mM potassium phosphate buffer, pH 7.4 ( appendix 2A) containing 0.25 M sucrose, 1 mM EDTA, and 1× protease inhibitor cocktail (Sigma)
  • Cotton wool
  • Small desiccator
  • 10‐ml syringe with needle
  • Surgical instruments
  • Potter‐Elvehjem homogenizer
  • 15‐ml centrifuge tubes
  • High‐speed refrigerated centrifuge
  • Bath sonicator
  • Dry ice‐ethanol mixture or liquid nitrogen

Support Protocol 2: Estimation of Protein Content of Mitochondrial Suspension

  Materials
  • Mitochondrial suspension ( protocol 2)
  • 0.1 N sodium hydroxide
  • 1 mg/ml bovine serum albumin stock solution in H 2O
  • Coomassie brilliant blue G‐250 working solution (see recipe)
  • 10‐ to 15‐ml glass test tubes
  • 80°C water bath
  • Spectrophotometer and glass cuvettes
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Figures

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Literature Cited

   Balaban, R.S., Brand, M.D., Affourtit, C., Esteves, T.C., Green, K., Lambert, A.J., Miwa, S., Pakay, J.L., and Parker, N. 2004. Mitochondria, oxidants and aging. Free Rad. Biol. Med. 37:755‐767.
   Balijepalli, S., Boyd, M.R., and Ravindranath, V. 1999. Inhibition of mitochondrial complex I by haloperidol: Role of thiol oxidation. Neuropharmacology 38:567‐577.
   Beer, S.M., Taylor, E.R., Brown, S.E., Dahm, C.C., Costa, N.J., Runswick, M.J., and Murphy, M.P. 2004. Glutaredoxin 2 catalyzes the reversible oxidation and glutathionylation of mitochondrial membrane thiol proteins: Implications for mitochondrial redox regulation and antioxidant defense. J. Biol. Chem. 279:47939‐47951.
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   Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of dye‐ binding. Anal. Biochem. 72:248‐254.
   Brandt, U. 2006. Energy converting NADH: Quinone oxidoreductase (Complex I). Annu. Rev. Biochem. 75:69‐92.
   Davey, G.P., Peuchen, S., and Clark, J.B. 1998. Energy thresholds in brain mitochondria: Potential involvement in neurodegeneration. J. Biol. Chem. 273:12753‐12757.
   Gellerich, F.N., Mayr, J.A., Reuter, S., Sperl, W., and Zierz, S. 2004. The problem of interlab variation in methods for mitochondrial disease diagnosis: Enzymatic measurement of respiratory chain complexes. Mitochondrion 4:427‐439.
   Haas, R.H., Nasirian, F., Nakano, K., Ward, D., Pay, M., Hill, R., and Shults, C.W. 1995. Low platelet mitochondrial complex I and complex II/III activity in early untreated Parkinson's disease. Ann. Neurol. 37:714‐722.
   Hirst, J., Carroll, J., Fearnley, I.M., Shannon, R.J., and Walker, J.E. 2003. The nuclear encoded subunits of complex I from bovine heart mitochondria. Biochim. Biophys. Acta 1604:135‐150.
   Jha, N., Jurma, O., Lalli, G., Liu, Y., Pettusi, E.H., Greenamyre, J.T., Liu, R.M., Forman, H.J., and Andersen, J.K. 2000. Glutathione depletion in PC12 results in selective inhibition of mitochondrial complex I activity. Implications for Parkinson's disease. J. Biol. Chem. 275:26096‐26101.
   Kenchappa, R.S. and Ravindranath, V. 2003. Glutaredoxin is essential for maintenance of brain mitochondrial complex I: Studies with MPTP. FASEB J. 17:717‐719.
   Kussmaul, L. and Hirst, J. 2006. The mechanism of superoxide production by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria. Proc. Natl. Acad. Sci. U.S.A. 103:7607‐7612.
   Li, Y., Huang, T.T., Carlson, E.J., Melov, S., Ursell, P.C., Olson, J.L., Noble, L.J., Yoshimura, M.P., Berger, C., Chan, P.H., Wallace, D.C., and Epstein, C.J. 1995. Dilated cardiomyopathy and neonatal lethality in mutant mice lacking manganese superoxide dismutase. Nat. Genet. 11:376‐381.
   Mann, V.M., Cooper, J.M., Krige, D., Daniel, S.E., Schapira, A.H.V., and Marsden, C.D. 1992. Brain, skeletal muscle and platelet homogenate mitochondrial function in Parkinson's disease. Brain 115:333‐342.
   Raha, S. and Robinson, B.H. 2000. Mitochondria, oxygen free radicals, disease and ageing. Trends Biochem. Sci. 25:502‐508.
   Schapira, A.H.V., Cooper, J.M., Dexter, D., Jenner, P., Clark, J.B., and Marsden, C.D. 1989. Mitochondrial complex I deficiency in Parkinson's disease. Lancet 1:1269.
   Shults, C.W., Nasirian, F., Ward, D.M., Nakano, K., Pay, M., Hill, R.R., and Haas, R.H. 1995. Cardidopa/Levodopa and selegiline do not affect platelet mitochondrial function in early Parkinsonism. Neurology 45:344‐348.
   Sriram, K., Shankar, S.K., Boyd, M.R., and Ravindranath, V. 1998. Thiol oxidation and loss of mitochondrial complex I precede excitatory amino acid mediated neurodegeneration. J. Neurosci. 18:10287‐10296.
   Taylor, E.R., Hurrell, F., Shannon, R.J., Lin, T.K., Hirst, J., and Murphy, M.P. 2003. Reversible glutathionylation of complex I increases mitochondrial superoxide formation. J. Biol. Chem. 278:19603‐19610.
   Tretter, L., Sipos, I., and Adam‐Vizi, V. 2004. Initiation of neuronal damage by complex I deficiency and oxidative stress in Parkinson's disease. Neurochem. Res. 29:569‐577.
   Turrens, J.F. 2003. Mitochondrial formation of reactive oxygen species. J. Physiol. 552:335‐344.
   Wallace, D.C. 1999. Mitochondrial diseases in man and mouse. Science 283:1482‐1488.
Key References
   Shults et al., 1995. See above.
  These two references provide detailed method for complex I activity estimation.
   Sriram et al., 1998. See above.
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