Isolation Purification and Separation Biochemistry

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Authors: Stefania Magri, Valentina Fracasso, Marco Rimoldi & Franco Taroni

Introduction

The aim of the protocol is to obtain an enriched fraction of intact mitochondria from the yeast Saccharomyces cerevisiae to perform quantitative determination of the activity of respiratory chain enzymes. All the strains used to perform this protocol are derivatives of W303 (K699: MATa ade2-1 trp1-1 can1-100 leu2-3,112 his3-11,15 ura3-52).

Reagents

Cell culture

  1. Selective medium (as appropriate) supplemented with 2% (w/v) D-glucose
  2. YPGAL medium: YEP medium (1% yeast extract, 2% Bactopeptone) supplemented with 2% (w/v) D-galactose and 0.1% (w/v) D-glucose

Preparation of mitochondria by differential centrifugations

  1. 10 mM ethylenediaminetetraacetic acid (EDTA)
  2. Sorbitol Buffer A: 1.2 M sorbitol, 50 mM Tris-HCl, pH7.5, 10 mM EDTA (stored at 4 °C), 0.3% (v/v) 2-mercaptoethanol added before use
  3. Sorbitol Buffer B: 0.7 M sorbitol, 50 mM Tris-HCl, pH 7.5, 0.2 mM EDTA (stored at 4 °C)
  4. Zymolyase-100T (Seikagaku Biobusiness Corporation) 4 mg/ml (stored at -20 °C)
  5. CompleteTM Protease Inhibitor Cocktail EDTA-free Tablets (Roche)

Sample preparation

  1. 10 mM potassium phosphate (PK) buffer, pH 7.4 (stored at -20 °C)
  2. Bradford microplate microassay (Bio-Rad)

Ubiquinol:cytochrome c oxidoreductase assay

  1. 250 mM potassium phosphate (PK) buffer, pH 7.4 (stored at -20 °C)
  2. 50 mM sodium azide (NaN3) (stored at -20 °C)
  3. 1 mM cytochrome c (freshly prepared)
  4. 1 mg/ml antimycin A in 50% ethanol (stored at -20 °C)
  5. sodium borohydride (NaBH4)
  6. decylubiquinone (DB)
  7. decylubiquinol (DBH2) (stored at 4 °C and freshly diluted 1:1 with H2O)

Cytochrome c oxidase assay

  1. 100 mM potassium phosphate (PK) buffer, pH 7.0 (stored at -20 °C)
  2. 0.8 mM cytochrome c (freshly prepared). Dissolve cytochrome c in H2O containing 10% of 100 mM potassium phosphate buffer, pH 7.0, and reduce it with sodium dithionite)
  3. 40 mM potassium cyanide (KCN) (stored at -20 °C)

ATP synthase assay

  1. Buffer H-Mg: 10 mM MgSO4 in 100 mM Hepes-KOH, pH 8.0 (stored at -20 °C)
  2. 30 mM NADH (freshly prepared)
  3. 50 mM phosphoenolpyruvic acid (freshly prepared)
  4. 10 mg/ml pyruvate kinase
  5. 5 mg/ml lactate dehydrogenase (stored at 4 °C)
  6. 0.2 mg/ml antimycin A in 50% ethanol (stored at -20 °C)
  7. 25 mM ATP buffered at pH 7.0 with 3M KHCO3 (freshly prepared)
  8. 0.2 mg/ml oligomycin in 50% ethanol (stored at -20 °C)

Equipment

  1. Shaking incubator at 28 °C
  2. Water bath at 37 °C
  3. Refrigerated centrifuges
  4. Spectrophotometer

Procedure

Cell culture

  1. Pre-inoculate yeast cells in 5 ml of liquid selective medium supplemented with 2% (w/v) glucose with shaking at 28 °C for 8h.
  2. Harvest cells and inoculate in 200 ml of YPGAL medium. Incubate overnight with shaking at 28 °C.
  3. Harvest cells by centrifuging when OD600nm of the culture reaches a value of 2.
  4. Wash the pellet in 20 ml of sterile H2O. The resulting pellet can be stored at -80 °C.

Preparation of mitochondria by differential centrifugations

Yeast cell mitochondria are prepared by differential centrifugations. This protocol is adapted from Ref. 1.

  1. Rinse the pellet with 5ml of 10 mM EDTA.
  2. Centrifuge at 400g for 3 min.
  3. Discard the supernatant and resuspend the pellet in 4.5 ml of ice-cold Sorbitol Buffer A supplemented with 0.3% (v/v) 2-mercaptoethanol.
  4. Digest the cell wall with approx. 250 µl of 4 mg/ml Zymolyase-100T (1 mg per g of cells) at 37 °C for 45 min.
  5. Verify wall digestion under the microscope.
  6. Harvest spheroplasts by centrifugation at 1,800g for 15 min at 4 °C.
  7. Discard the supernatant and resuspend the pellet gently in 7 ml of ice-cold Sorbitol Buffer B.
  8. Centrifuge at 2,500g for 15 min at 4 °C.
  9. Transfer the supernatant in a new tube and centrifuge at 20,000g for 15 min at 4 °C.
  10. Discard the supernatant and resuspend the pellet by pipetting in 4 ml of ice-cold Sorbitol Buffer B supplemented with EDTA-free CompleteTM Protease Inhibitor Cocktail (Roche).
  11. Centrifuge at 800g for 5 min at 4°C.
  12. Transfer the supernatant in a new tube and centrifuge at 15,000g for 15 min at 4 °C.
  13. Discard the supernatant and resuspend the pellet by pipetting in 4 ml of ice-cold Sorbitol Buffer B.
  14. Centrifuge at 800g for 5 min at 4°C.
  15. Transfer the supernatant in a new tube and centrifuge at 15,000g for 15 min at 4°C.
  16. Discard the supernatant and resuspend the pellet by pipetting in 1.5 ml of ice-cold Sorbitol Buffer B.
  17. Centrifuge at 800g for 5 min at 4°C.
  18. Decant the supernatant in a new 2-ml-tube and centrifuge at 15,000g for 15 min at 4°C.
  19. Store the pellet at -80°C.

Sample preparation

  1. Resuspend the resulting mitochondrial pellet in 300-500 µl of 10 mM PK buffer, pH 7.4.
  2. Freeze and thaw three times.
  3. Determine protein concentration with Bradford microplate microassay (Bio-Rad).
  4. Normalize samples for protein concentration (recommended concentration: between 0.3 and 0.6 µg/µl).

Ubiquinol:cytochrome c oxidoreductase (Complex III) activity assay

The protocol is adapted from Refs. 2 and 3.

A) Chemically reduce decylubiquinone (DB) to decylubiquinol (DBH2)

  1. Prepare a 10-mM solution of DB in HCl-acidified ethanol (pH 4 or lower).
  2. Add a few milligrams of sodium borohydride to reduce quinone. Reaction is completed when solution’s color changes from yellow to colorless.
  3. Add 2 ml of cyclohexane to the reduced solution and stir it.
  4. Transfer the upper (organic) phase to a clean tube.
  5. Add 2 ml of cyclohexane to the remaining lower phase and stir it. Transfer the upper phase adding it to the previous one.
  6. Repeat point 5 another time.
  7. Wash the organic fraction with 2 M NaCl.
  8. Dry the pellet under nitrogen and resuspend it with a volume of HCl-acidified ethanol (pH 4 or lower) corresponding to the initial one.

B) Determine ubiquinol:cytochrome c oxidoreductase activity

Activity is determined by measuring spectrophotometrically, at 550 nm at 30 °C, the rate of reduction of cytochrome c by ubiquinol. Measure complex III activity at several protein concentrations paying attention to the linearity of the reaction. To determine the reduction of cytochrome c due to the specific activity of complex III, for each sample, perform parallel measurements of activity in the presence and in the absence of antimycin A, an inhibitor of complex III.

  1. Prepare two cuvettes containing 200 µl of 250 mM PK buffer, pH 7.4, 40 µl of 50 mM NaN3, and 50 µl of 1 mM cytochrome c. Add 10 µl of 1 mg/ml antimycin A in one of them and an equal volume of H2O in the other one.
  2. Add 2-10 µg of mitochondrial proteins.
  3. Adjust the volume to 990 µl with H2O.
  4. Record the baseline for 2 min.
  5. Start the reaction by adding 10 µl of DBH2.
  6. Measure absorbance of the sample at 550 nm for 2 min.
  7. Calculate complex III specific activity using the Beer-Lambert law equation (Fig.1). Express the activity as nanomoles of cytochrome c reduced per minute per milligram of protein.

Cytochrome c oxidase (Complex IV) activity assay

Determine cytochrome c oxidase activity by measuring spectrophotometrically at 30 °C for 2 minutes the oxidation of cytochrome cred as indicated by the decrease of absorbance at 550 nm (Refs. 2-3). For each sample, measure complex IV activity twice at at least two protein concentrations that ensure the linearity of the reaction.

  1. Reduce cytochrome c by adding tiny amounts of sodium dithionite until the absorbance at 550 nm of 100 µl of cytochrome c in 1 ml of H2O is between 1.8 and 1.9.
  2. In a cuvette, add 100 µl of 100 mM PK buffer, pH 7.0, 100 µl of reduced cytochrome c and adjust the volume with H2O (800 µl minus sample volume).
  3. Incubate the cuvette at 30 °C for 2 min.
  4. Start the reaction by adding 2-10 µg of mitochondrial proteins.
  5. Measure absorbance of the sample at 550 nm for 2 min.
  6. Verify the specificity of cytochrome c reduction by inhibiting cytochrome c oxidase activity with 50 µl of 40 mM KCN.
  7. Calculate complex IV specific activity using the Beer-Lambert law equation (Fig. 1). Express the activity as nanomoles of cytochrome c oxidised per minute per milligram of protein.

ATP synthase (Complex V) activity assay

Determine complex V activity by measuring spectrophotometrically at 30 °C for 2 minutes the oxidation of NADH as indicated by the decrease of absorbance at 340 nm (Refs. 2-3). Repeat each measurement twice at at least two protein concentrations that ensure the linearity of the reaction.

  1. In a cuvette add 500 µl of Buffer H-Mg, 10 µl of 30 mM NADH, 50 µl of 50 mM phosphoenolpyruvic acid, 5 µl of 10 mg/ml of pyruvate kinase, 10 µl of 5 mg/ml of lactate dehydrogenase, and 10 µl of antimycin A.
  2. Add 2-10 µg of mitochondrial proteins.
  3. Adjust the volume to 900 µl with H2O.
  4. Incubate the cuvette at 30 °C for 2 minutes
  5. Record the baseline for 2 min.
  6. Start the reaction by adding 100 µl of 25 mM ATP.
  7. Measure absorbance of the sample for 2 min (?Abs340nmwithout oligomycin)
  8. Add 10 µl of oligomycin.
  9. Measure absorbance for 2min (?Abs340nmwith oligomycin).
  10. Calculate complex V specific activity using the Beer-Lambert law equation (Fig. 1). Express complex V activity as nanomoles of NADH oxidised per minute per milligram of protein.

Timing

3 days:

  • Cell culture: 8h (pre-culture in selective medium) + 20h (overnight culture in YPGAL)
  • Preparation of mitochondria by differential centrifugations: 4-6h
  • Sample preparation: 1h
  • Respiratory chain activity assay: variable, depending on the number of samples

Critical Steps

Preparation of mitochondria by differential centrifugations

  • Keep samples on ice during the entire procedure.
  • Following wall digestion, resuspend the pellets by gentle pipetting (not vortexing) to avoid mitochondria fragmentation.

References

  1. Lemaire, C. & Dujardin, G. Preparation of respiratory chain complexes from Saccharomyces cerevisiae wild-type and mutant mitochondria: activity measurement and subunit composition analysis. Methods Mol Biol 432, 65-81 (2008).
  2. Rimoldi, M. et al. Cytochrome-c-oxidase deficiency in muscles of a floppy infant without mitochondrial myopathy. J Neurol 227, 201-207 (1982).
  3. Dionisi-Vici, C. et al. Fulminant Leigh syndrome and sudden unexpected death in a family with the T9176C mutation of the mitochondrial ATPase 6 gene. J. Inherit Metab Dis 21, 2-8 (1998).

Figures

Figure 1: Formulae to calculate Complex III, Complex IV, and Complex V activity

Fig 1

Formulae are derived from the Beer-Lambert law equation. Activities are expressed as nanomoles per minute per milligram of protein.

Associated Publications

Mutations in the mitochondrial protease gene AFG3L2 cause dominant hereditary ataxia SCA28, Daniela Di Bella, Federico Lazzaro, Alfredo Brusco, Massimo Plumari, Giorgio Battaglia, Annalisa Pastore, Adele Finardi, Claudia Cagnoli, Filippo Tempia, Marina Frontali, Liana Veneziano, Tiziana Sacco, Enrica Boda, Alessandro Brussino, Florian Bonn, Barbara Castellotti, Silvia Baratta, Caterina Mariotti, Cinzia Gellera, Valentina Fracasso, Stefania Magri, Thomas Langer, Paolo Plevani, Stefano Di Donato, Marco Muzi-Falconi, and Franco Taroni, Nature Genetics 42 (4) 313 - 321 07/03/2010 doi:10.1038/ng.544

Author information

Stefania Magri, Valentina Fracasso, Marco Rimoldi & Franco Taroni, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy

Source: Protocol Exchange (2010) doi:10.1038/nprot.2010.25. Originally published online 7 March 2010.

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