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Determination of the H+-ATP Synthase and Hydrolytic Activities
测定 H+-ATP 合成酶的合成和水解活性   

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Abstract

The H+-ATP synthase of the inner mitochondrial membrane utilizes the proton gradient generated by the respiratory chain to synthesize ATP. Under depolarizing conditions it can function in reverse by hydrolyzing ATP to generate a proton gradient. The protocols presented here allow the facile determination of both the synthetic and hydrolytic activities of the H+-ATP synthase in isolated mitochondria and in permeabilized mammalian cells. Since the protocol requires the isolation of polarized and well-coupled mitochondria, first we describe the protocol for mitochondrial isolation from mouse tissues. Second, we describe the protocol for measuring the ATP synthetic activity as end-point and kinetic modes in isolated mitochondria and in permeabilized cells. Finally, we describe the protocol for the determination of the ATP hydrolytic activity of the enzyme in isolated mitochondria.

Keywords: Mitochondria(线粒体), Oxidative phosphorylation(氧化磷酸化), Enzyme activity(酶的活性), ATP synthase(ATP合成酶), ATP hydrolase(ATP水解酶)


Part I. The isolation of mitochondria from mouse tissues

Materials and Reagents

  1. 10 ml tubes
  2. 1.5 ml Eppendorf tubes
  3. Sucrose (Merck Millipore Corporation, catalog number: 107651 )
  4. Ethylenediaminetetraacetic acid disodium salt dehydrate (Sigma-Aldrich, catalog number: ED2SS )
  5. D-Sorbitol (Sigma-Aldrich, catalog number: S1876 )
  6. Ethylene glycol-bis (2-aminoethylether)-N,N,N′,N′-tetraacetic acid (Sigma-Aldrich, catalog number: E4378 )
  7. Trizma® base (Sigma-Aldrich, catalog number: T1503 )
  8. Bovine serum albumin (Sigma-Aldrich, catalog number: A7906 )
  9. Protein assay dye reagent concentrate (Bio-Rad Laboratories, catalog number: 5000006 )
  10. Medium A (see Recipes)
  11. Medium H (see Recipes)

Equipment

  1. 15 ml Dounce All-Glass tissue grinder (Kimble, catalog number: 885303-0015 )
  2. Beckman Avanti J-25 centrifuge (Beckman Coulter, model: Avanti J-25 )
  3. JA-25.50 rotor, fixed angle, aluminum, single lock lid, 8 x 50 ml, 25,000 rpm, 75,600 x g (Beckman Coulter, catalog number: 363055 )
  4. Bio-VialTM tube, PP, 4 ml, 14 x 55 mm, 0.56 x 2.2 in (Beckman Coulter, catalog number: 566353 )
  5. Centrifuge 5415R (Sigma-Aldrich, Eppendorf®, model: 5415R )

Procedure

  1. Animal studies were carried out in compliance with animal policies and ethical guidelines of the European Community. The project was approved by the Institutional Review Board (Ethical Committee of the UAM, CEI-24-571).
  2. The isolation of mitochondria from mouse liver and/or heart was performed according to (Fernandez-Vizarra et al., 2010) with minor modifications.
  3. Adult mice are sacrificed by cervical dislocation and the liver (roughly half of the liver without the gallbladder) or the whole heart is removed and immediately cooled down at 4 °C in the homogenization medium A (liver) or medium H (heart). At this point, place several 10 ml centrifuge tubes on ice until step 6.
  4. The tissue is washed, weighted and minced into small pieces with a pair of scissors in precooled 1x PBS to remove blood and connective tissue.
  5. The homogenization is performed in the tissue grinder with 4 ml/g of cold homogenization medium A (liver) or with 10 ml/g of cold homogenization medium H (heart). It is necessary to make 5 steps with pestle A followed by 5 steps with pestle B in the case of liver and 12 steps with pestle A followed by 12 steps with pestle B in the case of heart.
  6. The homogenate is transferred to the precooled 10 ml tubes and centrifuged for 10 min at 1,000 x g at 4 °C. This step is then repeated with the supernatant in order to discard unbroken tissue, cells and nuclei.
  7. The resulting supernatant is transferred to 1.5 ml Eppendorf tubes and centrifuged in a microfuge for 10 min at 6,700 x g at 4 °C to pellet the mitochondria.

Notes

  1. It is very important to perform the entire isolation process at 4 °C using cold medium and without long intermediate stops.
  2. Mitochondria should not be centrifuged any more in order to preserve functional integrity of the organelle.

Recipes

  1. Medium A (100 ml)
    0.32 M sucrose (MW = 342.2965 g/mol): 10.95 g
    1 mM EDTA (MW = 292.24 g/mol): 30 mg
    10 mM Tris-HCl (pH 7.4) (MW = 121.14 g/mol): 0.12 g
  2. Medium H (100 ml)
    0.072 M sucrose (MW = 342.2965 g/mol): 2.46 g
    0.225 M sorbitol (MW = 182.17 g/mol): 4.1 g
    1 mM EGTA (MW = 380.4 g/mol): 40 mg
    0.1% w/v fatty acid-free bovine serum albumin (BSA): 0.1 g
    10 mM Tris-HCl (pH 7.4) (MW = 121.14 g/mol): 0.12 g

Part II. Determination of the ATP synthetic activity of the H+-ATP synthase

Materials and Reagents

  1. Microplate, 96 well, Polystyrene, black flat bottom, TC-treated (Cultek, catalog number: 153603 )
  2. Protein assay dye reagent concentrate (Bio-Rad Laboratories, catalog number: 5000006 )
  3. Phosphatase inhibitor cocktail 2 (Sigma-Aldrich, catalog number: P5726 )
  4. cOmpleteTM, Mini, EDTA-free (Sigma-Aldrich, Roche Diagnostics®, catalog number: 11836170001 )
  5. Digitonin (Sigma-Aldrich, catalog number: D141 )
  6. P1, P5-di(adenosine-5′) pentaphosphate pentasodium salt (Sigma-Aldrich, catalog number: D4022 )
  7. Adenosine 5′-diphosphate sodium salt (Sigma-Aldrich, catalog number: A2754 )
  8. Adenosine 5′-triphosphate (ATP) disodium salt hydrate (Sigma-Aldrich, catalog number: FLAAS )
  9. HClO4
  10. KOH
  11. Oligomycin (VWR International, catalog number: 80058-538 )
  12. Succinic acid (Sigma-Aldrich, catalog number: 398055 )
  13. Rotenone (Sigma-Aldrich, catalog number: R8875 )
  14. ATP bioluminescence assay kit CLS II (Roche Diagnostics, catalog number: 11699695001 )
  15. Luciferase from Photinus pyralis (Sigma-Aldrich, catalog number: 10411523001 )
  16. Beetle luciferin, potassium salt (Promega, catalog number: E1601 )
  17. pH indicator solution (pH 4.0-10.0) (Merck Millipore Corporation, catalog number: 1091750100 )
  18. Trizma® base (Sigma-Aldrich, catalog number: T1503 )
  19. Bovine serum albumin (Sigma-Aldrich, catalog number: A7906 )
  20. Sucrose (Merck Millipore Corporation, catalog number: 107651 )
  21. KCl (Merck Millipore Corporation, catalog number: 104936 )
  22. MgCl2 (Merck Millipore Corporation, catalog number: 172571 )
  23. Phosphate-potassium buffer (Sigma-Aldrich, catalog number: P0662 / P3786 )
  24. Ethylene glycol-bis (2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) (Sigma Aldrich, catalog number: E4378 )
  25. Respiration Medium (RM) (see Recipes)

Equipment

  1. FLUOstar Omega Microplate Reader (BMG Labtech, model: FLUOstar Omega )
  2. Heraeus Megafuge 11R (Thermo Fisher Scientific, model: Heraeus Megafuge 11R )
  3. Centrifuge 5415R (Sigma-Aldrich, Eppendorf®, model: 5415R )

Procedure

  1. End Point determination of the ATP synthetic activity
    1. If working with isolated mitochondria from animal tissues the isolated organelles are resuspended at 1 µg/µl of protein in Respiration Medium (RM) containing 1x phosphatase and protease inhibitors. The mitochondrial preparation is kept at 4 °C. No further procedures are required until step A7.
    2. If working with cells, the cells (~1-3 x 106 cells) are trypsinized and washed twice with PBS and the cellular pellet resuspended in 1 ml of RM at RT containing 1x phosphatase and protease inhibitors. Cellular permeabilization is achieved by addition of digitonin at a concentration of 50-75 µg/ml of RM.
    3. Tubes are shaken by repeated inversions during 1 min. For the removal of digitonin, immediate centrifugation of the cells at 100 x g during 5 min followed by two washes with RM at room temperature is required.
    4. At this point, a small fraction of the permeabilized cells are separated and used for the quantification of total protein concentration using a colorimetric based assay such as Bradford Protein Assay or the Bicinchoninic acid Assay (BCA).
    5. Prepare two sets of tubes; one is needed for the reaction without oligomycin (OL) and one for the parallel reaction with oligomycin. In addition, prepare two sets of seven tubes each containing 200 μl of 6% HClO4 to be used under step A9.
    6. RM is used for the preparation of the reaction buffer by supplementation with 150 µM P1, P5-di(adenosine-5′) pentaphosphate (an adenylate kinase inhibitor that prevents conversion of ADP into ATP), 2 mM rotenone, 5 mM ADP and 2 mM succinate as respiratory substrate (Complex II). Other respiratory substrates can be used.
    7. The parallel reaction using reaction buffer supplemented with 30 µM OL is assayed for each sample. This reaction indicates the ATP synthesized that might occur independent of the H+-ATP synthase activity.
    8. Triggering of the reaction of ATP synthesis is achieved by the addition of 150-200 μg of protein of permeabilized cells or of 50 μg of the isolated mitochondria to the tube containing 450 μl of the reaction buffer (as described under steps A6 and A7) in a tube shaker at constant shaking and at 30 °C.
    9. Small aliquots (50 μl) are taken every 30 sec until 3 min (end of reaction) and added to the tubes containing 200 μl of 6% HClO4.
    10. The tubes are immediately vortexed and placed at 4 °C during 1 h for the precipitation of proteins.
    11. Afterwards, tubes are centrifuged at 11,000 x g during 5 min and the resulting supernatant transferred to another tube for neutralization with 10% KOH (~130 µl) in the presence of 2 µl of pH indicator.
    12. The content of ATP in each sample is determined using the ATP bioluminescence assay kit CLS II in 96-well plates with a luminometer plate-reader. To this aim, mix in each well 50 μl of the neutralized sample with 50 μl of the reaction buffer included in the kit and containing luciferase. An ATP standard curve (0-10 µM) is generated and processed in the same plate adding 50 μl of ATP standard and 50 μl of the reaction buffer included in the kit.
    13. The initial rate of ATP synthesis is determined by estimation of the pmoles of ATP produced in each sample. The ATP synthetic activity of the enzyme is expressed as nmoles of ATP/min/mg of protein (as indicated in Representative data, Figure 1).

  2. Kinetic determination of the ATP synthetic activity
    1. For assaying the mitochondrial ATP synthetic activity in kinetic mode we followed the protocol detailed in (Vives-Bauza et al., 2007) with some modifications.
    2. Steps B1-4 were performed according to the previous protocol.
    3. Permeabilized cells or 50-150 µg of isolated mitochondria are resuspended in 40 µl of RM. The tubes are kept on ice until used.
    4. Two mixes A and B with the reaction compounds are prepared in RM (see Recipes 2 and 3)
    5. Add 160 µl of Mix A with 20 µl of Mix B in each well. The luminometer plate-reader is set up to measure luminescence in kinetic mode every 10 sec during ~10 min in which luminescence increases linearly for ~250 sec.
    6. Place the plate in the luminometer and add 20 µl of permeabilized cells or isolated mitochondria obtained in step B3 to start the reaction of ATP synthesis.
    7. Relative light units are converted to ATP concentration using an ATP standard curve. To this end, prepare triplicates of ATP solutions between 0 and 10 µM in Mix A at a final volume of 180 µl. Finally, add 20 µl of Mix B to each well and measure luminescence at end point (see Figure 2).
    8. The initial rate of ATP synthesis is determined by estimation of the pmoles of ATP produced in each sample. The ATP synthetic activity of the enzyme is expressed as pmoles of ATP/min/mg of protein (as indicated in Representative data, Figure 2).

Representative data

  1. To convert the RLU obtained into ATP produced it is necessary to follow the next steps:
    1. Subtract the RLU in T0, T1, T2 and T3 time points in tubes + OL (a relatively high value because of the ATP contained in mitochondria) from the time points taken in the absence of OL (T0, T1, T2, T3) (Figure 1A). It is very important to verify that RLU increases linearly and significantly in the absence of OL (Figure 1A) whereas subtle or negligible changes in RLU occur in the presence of OL (Figure 1A).
    2. Interpolate the RLU values obtained (Figure 1A) in the linear regression (Figure 1B) to obtain the ATP content in the volume of sample that has been measured.
    3. Multiply the pmol of ATP obtained by the fixed value of 7.6, which is the ratio between the total volume and the volume of sample measured. (Total volume = 250 µl + 130 µl = 380 µl); (Volume of sample = 50 µl); (Ratio = 7.6) (from steps A9 and A11).
    4. Multiply the value obtained under point 3 by the factor that indicates the volume of sample taken at each time point relative to the total volume of reaction left (i.e., Factor 9 at T1 because 450/50 = 9; Factor 8 at T2 because 400/50 = 8; Factor 7 at T3 because 350/50 = 7, ….) (from step A9).
    5. Normalized the total pmol of ATP obtained by the protein amount and reaction time (Figure 1C).
    6. The same calculation procedure is followed when the activity of a permeabilized-cell preparation is used.


      Figure 1. ATP synthase activity (end point mode) in isolated mitochondria from mouse heart in the absence (-OL) or presence (+OL) of oligomycin (OL). A. Relative light units (RLU) of samples at 0, 1, 2 and 3 min after triggering the reaction in the absence and presence of OL. B. Linear regression analysis for the correlation between ATP amount and luciferin/luciferase-dependent luminescence. C. Example of the ATP synthetic activity (mean ± S.E.M) using three preparations of heart mitochondria.

  2. To convert the RLU obtained into ATP produced it is necessary to follow the next steps:
    1. Subtract the RLU at T0 from all other time points (Figure 2A).
    2. To convert the RLU of the reaction into ATP amount it is necessary to interpolate the RLU at 60 sec in the linear regression (Figure 2B).
    3. It is important to normalized data according to the protein amount in the sample (Figure 2C).
      Note: If you use the RLU at 60 sec to interpolate into Figure 2B, your data is already a rate (pmol ATP/min).
    4. The same calculation procedure is followed when the activity of a preparation of isolated mitochondria is used.


      Figure 2. ATP synthase activity (kinetic mode) in digitonin-permeabilized HCT116 cells in the absence (blue) or presence of oligomycin (pink). A. Kinetic representation of the production of ATP in relative light units (RLU). The gray dotted line represents the initial rate of the phosphorylation reaction of ADP that is linear for at least 60 sec and it can be calculated from the slope of the line. B. Linear regression analysis for the correlation between ATP amount and luciferin/luciferase-dependent luminescence. C. Example of the ATP synthetic activity (mean ± S.E.M.) using three preparations of digitonin-permeabilized HCT116 cells.

Notes

  1. The concentration of digitonin and time of permeabilization are critical and might change depending on the cell line. Therefore, we recommend the previous titration of the digitonin concentration to be used in each cell line that better fits the determination of the ATP synthetic activity.
  2. It is recommended to prepare ADP freshly before use.

Recipes

  1. Respiration Medium (RM) (100 ml)
    225 mM sucrose (MW = 342.2965 g/mol): 7.7 g
    10 mM KCl (MW = 74.5 g/mol): 74 mg
    5 mM MgCl2 (MW = 95.2 g/mol): 47.6 mg
    0.05 % w/v bovine serum albumin: 50 mg
    10 mM potassium-phosphate buffer [HK2PO4 (MW = 174.17 g/mol): 0.17 g; H2KPO4 (MW = 136.1 g/mol): 0.16 g]
    1 mM EGTA (MW = 380.35 g/mol): 38 mg
    10 mM Tris-HCl (pH 7.4) (MW = 121.14 g/mol): 38 mg
  2. Mix A
    Respiration Medium (RM)
    0.1 mM ADP
    5 mM succinate
    0.15 µM P1, P5-di(adenosine-5′) pentaphosphate
    2 µg/ml rotenone with or without 30 µM oligomycin
  3. Mix B
    RM
    0.25 mg/ml of luciferin
    0.02 mg/ml luciferase.

Part III. Determination of the ATP hydrolytic activity of the H+-ATP synthase

Materials and Reagents

  1. Microplate, 96 well, Polystyrene, black flat bottom, TC-treated (Cultek, catalog number: 153603 )
  2. Protein assay dye reagent concentrate (Bio-Rad Laboratories, catalog number: 5000006 )
  3. Liquid nitrogen
  4. Phosphatase inhibitor cocktail 2 (Sigma-Aldrich, catalog number: P5726 )
  5. cOmpleteTM, Mini, EDTA-free (Sigma-Aldrich, Roche Diagnostics®, catalog number: 11836170001 )
  6. Oligomycin (VWR International, catalog number: 80058-538 )
  7. Trizma® base (Sigma-Aldrich, catalog number: T1503 )
  8. Bovine serum albumin (Sigma-Aldrich, catalog number: A7906 )
  9. Adenosine 5′-triphosphate (ATP) disodium salt hydrate (Sigma-Aldrich, catalog number: FLAAS )
  10. KCl (Merck Millipore Corporation, catalog number: 104936 )
  11. MgCl2 (Merck Millipore Corporation, catalog number: 172571 )
  12. Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) (Sigma-Aldrich, catalog number: C2920 )
  13. Antimycin A (Sigma-Aldrich, catalog number: A8674 )
  14. Phospho(enol)pyruvic acid cyclohexylammonium salt (Sigma-Aldrich, catalog number: P3637 )
  15. L-Lactate dehydrogenase (L-LDH) from rabbit muscle (Sigma-Aldrich, Roche Diagnostics®, catalog number: 10127876001 )
  16. Pyruvate kinase from rabbit muscle (Sigma-Aldrich, catalog number: P9136 )
  17. β-Nicotinamide adenine dinucleotide, reduced dipotassium salt (NADH) (Sigma-Aldrich, catalog number: N4505 )
  18. Reaction Buffer (RB) stock solution (see Recipes)
  19. FCCP stock solution (see Recipes)
  20. Antimycin A stock solution (see Recipes)
  21. Complete reaction buffer (see Recipes)
    1. 10 mM PEP stock solution
    2. 2.5 mM ATP stock solution
    3. 1 mM NADH stock solution
    4. Oligomycin A stock solution

Equipment

  1. FLUOstar Omega Microplate Reader (BMG Labtech, model: FLUOstar Omega )
  2. Heraeus Megafuge 11R (Thermo Fisher Scientific, model: Heraeus Megafuge 11R )
  3. Centrifuge 5415R (Sigma-Aldrich, Eppendorf®, model: 5415R )

Procedure

  1. ATP hydrolysis by the enzyme can be determined spectrophotometrically following the changes in Absorbance at 340 nm (A340). The basis of this method is the coupling of the enzymatic reactions catalyzed by lactate dehydrogenase (LDH) and pyruvate kinase (PK) to the hydrolytic activity of the ATP synthase (Barrientos et al., 2009). The ADP generated by the hydrolysis of ATP allows PK to transform 1 mole of phosphoenolpyruvate (PEP) into 1 mole of pyruvate. Pyruvate is then used by LDH to generate lactate oxidizing a molecule of NADH to NAD+ in the process. By each ATP molecule hydrolyzed one molecule of NADH is oxidized, allowing the detection of the reaction by a diminution in A340.
  2. Determination of the hydrolytic activity of the H+-ATP synthase is assayed on isolated broken mitochondria by three cycles of freezing and thawing the mitochondrial preparation in liquid nitrogen and at 37 °C, respectively.
  3. 30-50 µg of isolated mitochondria are resuspended in 20 µl of Reaction Buffer (RB) with 1x phosphatase and protease inhibitors.
  4. The luminometer plate-reader is set up to measure absorbance at 340 nm in kinetic mode (10 flashes per well) and the change in the absorbance is measured through constant intervals of time (15 sec). With these conditions it is possible to measure 24 samples simultaneously.
  5. 80 µl of complete RB (containing PK and LDH) and 20 µl of the mitochondria prepared as on step 3 are added in each 96 well black clear bottom plate and the reaction is recorded.
  6. When enough ATP hydrolysis has been produced (2-5 min), 30 µM oligomycin is added to each well. The addition of oligomycin inhibits the activity of the mitochondrial ATP synthase/hydrolase providing confirmation of the specificity of the assay.
  7. The ATPase activity of isolated mitochondria is expressed in nanomoles of NADH oxidized/min/mg of mitochondrial protein which is calculated using the Lambert-Beer equation and a molar extinction coefficient of NADH of 6.22 x 103 M-1 cm-1.
  8. N = ΔA340/(ε340 x l) where N is the concentration of NADH oxidized, ΔA340 is the decrement in absorbance between two given time-points, ε340 is the Molar extinction coefficient of NADH at 340 nm and l is the light path length of 1 cm.

Representative data



Figure 3. ATP hydrolase activity in isolated mitochondria from mouse heart. The slope of the graphs indicates the decrement of A340 as a function of reaction time. Where indicated, 30 μM of oligomycin (+OL, arrowhead) was added. Two different preparations containing 60 μg (A, red) and 30 μg (B, blue) of isolated mitochondria were assayed. Addition of OL (closed bar) blocks the hydrolase activity as evidence by the suppression of slope of the reaction. The ATP hydrolase activity is expressed as mU/mg protein. Bars represent the mean ± S.E.M. of the ATPase activity determined in three preparations of isolated mitochondria from mouse hearts.

Notes

  1. Note that the RB added at step 3 is not supplemented with PK and LDH.
  2. ATP and NADH must be prepared freshly.
  3. The FCCP, antimycin A, PEP and oligomycin stock solutions can be store at -20 °C.   

Recipes

  1. Reaction Buffer (RB) stock solution (10 ml)
    50 mM Tris-HCl (pH 8.0) (MW = 121.14 g/mol): 61 mg
    5 mg/ml bovine serum albumin: 50 mg
    20 mM MgCl2 (MW = 95.2 g/mol): 19 mg
    50 mM KCl (MW = 74.55 g/mol): 37.2 mg
  2. 2.5 mM FCCP stock solution
    FCCP (MW = 254.17 g/mol)
    Dissolve 0.6 mg FCCP in 1 ml of DMSO
  3. 500 µM antimycin A stock solution
    Antimycin A ‎(MW = 548.63 g/mol)
    Dissolve 0.3 mg antimycin A in 1 ml of ethanol
  4. Complete Reaction Buffer, 1 ml (enough volume of RB to run ten assays)
    1. 0.92 ml RB stock solution
    2. 2 µl 2.5 mM FCCP stock solution (Final concentration: 5 μM)
    3. 2 µl 500 µM antimycin A stock solution (Final concentration: 1 μM)
    4. 10 µl 1 M PEP (Final concentration: 10 μM)
      Dissolve 27 mg PEP in 100 μl of water
    5. 25 µl 100 mM ATP (Final concentration: 2.5 mM)
      Dissolve 5.5 mg in 100 µl of water
    6. 10 µl 100 mM NADH (Final concentration: 1 mM)
      Dissolve 7.4 mg in 100 µl of water
      13 µl 4 units of LDH (2,750 U/ml)
      20 µl 4 units of PK (1,920 U/ml)
      Note: The addition of 4 units of LDH and of 4 units of PK is done in the last step and just before triggering the reaction by the addition of mitochondria.
    7. 2 µl 1.5 mM Oligomycin A stock solution
      Dissolve 1.2 mg oligomycin stock solution in 1 ml of ethanol

Acknowledgments

We thank Drs. María Sánchez-Aragó and Laura Formentini for expert guidance in setting the assays of the ATP synthase activities. The technical assistance of M. Chamorro and C. Nuñez de Arenas is acknowledged. The authors are grateful to the previous work developed in the labs of Drs. Enríquez, Barrientos and Manfredi for developing the methods adapted in these protocols. JGB and CNT were supported by pre-doctoral fellowships from FPI-MICINN/MINECO and Fondo Social Europeo, Spain. This work was supported by grants from Ministerio de Economía y Competitividad (SAF2013-41945-R), Comunidad de Madrid (S2011/BMD-2402), and Fundación Ramón Areces (FRA), Spain. The CBMSO receives an institutional grant from the FRA.

References

  1. Barrientos, A., Fontanesi, F. and Diaz, F. (2009). Evaluation of the mitochondrial respiratory chain and oxidative phosphorylation system using polarography and spectrophotometric enzyme assays. Curr Protoc Hum Genet Chapter 19: Unit19 13.
  2. Fernandez-Vizarra, E., Ferrin, G., Perez-Martos, A., Fernandez-Silva, P., Zeviani, M. and Enriquez, J. A. (2010). Isolation of mitochondria for biogenetical studies: An update. Mitochondrion 10(3): 253-262.
  3. Vives-Bauza, C., Yang, L. and Manfredi, G. (2007). Assay of mitochondrial ATP synthesis in animal cells and tissues. Methods Cell Biol 80: 155-171.

简介

内线粒体膜的H sup + -ATP合酶利用呼吸链产生的质子梯度来合成ATP。 在去极化条件下,它可以通过水解ATP产生质子梯度而反向作用。 本文提供的方案允许容易地测定分离的线粒体和透化的哺乳动物细胞中H sup + -ATP合酶的合成和水解活性。 由于该协议需要极化和良好耦合的线粒体的分离,首先我们描述线粒体从小鼠组织分离的协议。 第二,我们描述了用于测量ATP合成活性作为终点和在分离的线粒体和透化细胞中的动力学模式的方案。 最后,我们描述了用于测定酶在分离的线粒体中的ATP水解活性的方案。

关键字:线粒体, 氧化磷酸化, 酶的活性, ATP合成酶, ATP水解酶

第I部分。线粒体与小鼠组织的分离

材料和试剂

  1. 10ml管
  2. 1.5 ml Eppendorf管
  3. 蔗糖(Merck Millipore Corporation,目录号:107651)
  4. 脱水的乙二胺四乙酸二钠盐(Sigma-Aldrich,目录号:ED2SS)
  5. D-山梨醇(Sigma-Aldrich,目录号:S1876)
  6. 乙二醇 - 双(2-氨基乙醚)-N,N,N',N'-四乙酸(Sigma-Aldrich,目录号:E4378)
  7. (Sigma-Aldrich,目录号:T1503)
  8. 牛血清白蛋白(Sigma-Aldrich,目录号:A7906)
  9. 蛋白质测定染料试剂浓缩物(Bio-Rad Laboratories,目录号:5000006)
  10. 中A(参见配方)
  11. 中等(见配方)

设备

  1. 15ml Dounce全玻璃组织研磨机(Kimble,目录号:885303-0015)
  2. Beckman Avanti J-25离心机(Beckman Coulter,型号:Avanti J-25)
  3. JA-25.50转子,固定角度,铝,单锁盖,8×50ml,25,000rpm,75,600×g(Beckman Coulter,目录号:363055)
  4. Bio-Vial TM管,PP,4ml,14×55mm,0.56×2.2英寸(Beckman Coulter,目录号:566353)
  5. 离心机5415R(Sigma-Aldrich,Eppendorf ,型号:5415R)

程序

  1. 动物研究是根据欧洲共同体的动物政策和伦理准则进行的。 该项目由机构审查委员会(UAM的伦理委员会,CEI-24-571)批准。
  2. 从小鼠肝和/或心脏分离线粒体根据(Fernandez-Vizarra等人,2010)进行微小修改。
  3. 通过颈脱位法处死成年小鼠,取出肝脏(大约一半没有胆囊的肝脏)或全心脏,并立即在均质化培养基A(肝脏)或培养基H(心脏)中在4℃冷却。 此时,将几个10ml离心管置于冰上,直到步骤6
  4. 用预冷的1×PBS中的一把剪刀将组织洗涤,称重并切成小块,以除去血液和结缔组织。
  5. 在组织研磨机中用4ml/g冷匀浆培养基A(肝)或用10ml/g冷匀浆培养基H(心脏)进行匀浆。有必要用杵A进行5步,接着用肝杵用杵B进行5步,用杵A进行12步,然后用心杵用杵B进行12步。
  6. 将匀浆转移至预冷的10ml管中,并在4℃以1,000xg离心10分钟。然后用上清液重复该步骤,以便丢弃未破损的组织,细胞和细胞核
  7. 将所得上清液转移至1.5ml Eppendorf管中,并在微量离心机中在4℃下以6,700×g离心10分钟以沉淀线粒体。

笔记

  1. 使用冷介质并且没有长时间的中间停止,在4℃下进行整个分离过程是非常重要的
  2. 线粒体不应再进行离心以保持细胞器的功能完整性。

食谱

  1. 中等A(100 ml)
    0.32M蔗糖(MW = 342.2965g/mol):10.95g
    1mM EDTA(MW = 292.24g/mol):30mg
    10mM Tris-HCl(pH7.4)(MW = 121.14g/mol):0.12g
  2. 中H(100 ml)
    0.072M蔗糖(MW = 342.2965g/mol):2.46g
    1mM EGTA(MW = 380.4g/mol):40mg
    0.1%w/v无脂肪酸的牛血清白蛋白(BSA):0.1g/mL 10mM Tris-HCl(pH7.4)(MW = 121.14g/mol):0.12g
第二部分。 确定H的ATP合成活性 + span style ="color:#666666; font-family:Arial; font-size:14px;"> - ATP合成器

材料和试剂

  1. 微孔板,96孔,聚苯乙烯,黑色平底,TC处理(Cultek,目录号:153603)
  2. 蛋白质测定染料试剂浓缩物(Bio-Rad Laboratories,目录号:5000006)
  3. 磷酸酶抑制剂混合物2(Sigma-Aldrich,目录号:P5726)
  4. (Sigma-Aldrich,Roche Diagnostics ,目录号:11836170001)的微量培养基
  5. Digitonin(Sigma-Aldrich,目录号:D141)
  6. (腺苷-5')五磷酸盐五钠盐(Sigma-Aldrich,目录号:D4022),其中p
  7. 腺苷5'-二磷酸钠盐(Sigma-Aldrich,目录号:A2754)
  8. 腺苷5'-三磷酸(ATP)二钠盐水合物(Sigma-Aldrich,目录号:FLAAS)
  9. HClO 4
  10. KOH
  11. 寡霉素(VWR International,目录号:80058-538)
  12. 琥珀酸(Sigma-Aldrich,目录号:398055)
  13. Rotenone(Sigma-Aldrich,目录号:R8875)
  14. ATP生物发光测定试剂盒CLS II(Roche Diagnostics,目录号:11699695001)
  15. 来自萤火虫的萤光素酶(Sigma-Aldrich,目录号:10411523001)
  16. 甲虫荧光素,钾盐(Promega,目录号:E1601)
  17. pH指示剂溶液(pH 4.0-10.0)(Merck Millipore Corporation,目录号:1091750100)
  18. (Sigma-Aldrich,目录号:T1503)
  19. 牛血清白蛋白(Sigma-Aldrich,目录号:A7906)
  20. 蔗糖(Merck Millipore Corporation,目录号:107651)
  21. KCl(Merck Millipore Corporation,目录号:104936)
  22. MgCl 2(Merck Millipore Corporation,目录号:172571)
  23. 磷酸钾缓冲液(Sigma-Aldrich,目录号:P0662/P3786)
  24. 乙二醇 - 双(2-氨基乙醚)-N,N,N',N'-四乙酸(EGTA)(Sigma Aldrich,目录号:E4378)
  25. 呼吸中(RM)(见配方)

设备

  1. FLUOstar Omega微孔板读数器(BMG Labtech,型号:FLUOstar Omega)
  2. Heraeus Megafuge 11R(Thermo Fisher Scientific,型号:Heraeus Megafuge 11R)
  3. 离心机5415R(Sigma-Aldrich,Eppendorf ,型号:5415R)

程序

  1. 终点确定ATP合成活性
    1. 如果与来自动物组织的分离的线粒体一起工作,将分离的细胞器以1μg/μl的蛋白质重悬于含有1×磷酸酶和蛋白酶抑制剂的呼吸培养基(RM)中。线粒体制剂保持在4℃。直到步骤A7,不需要进一步的程序
    2. 如果与细胞一起工作,将细胞(约1-3×10 6个细胞)胰蛋白酶化并用PBS洗涤两次,并将细胞沉淀物重悬浮于含有1×磷酸酶和蛋白酶抑制剂的1ml RM中。通过添加浓度为50-75μg/ml RM的毛地黄皂苷实现细胞透化。
    3. 通过重复倒置在1分钟期间摇动管。为了去除毛地黄皂苷,需要在100分钟内立即离心细胞5分钟,然后在室温下用RM洗涤两次。
    4. 在这一点上,分离小部分透化细胞,并使用基于比色的测定法例如Bradford蛋白质测定法或二辛可宁酸测定法(BCA)用于总蛋白质浓度的定量。
    5. 准备两套管;需要一个用于没有寡霉素(OL)的反应,一个用于与寡霉素的平行反应。此外,制备两组七个管,每个管含有200μl的在步骤A9下使用的6%HClO 4。
    6. RM用于通过补充150μM的P 1,P 5 - 二(腺苷-5')五磷酸(腺苷酸激酶抑制剂)来制备反应缓冲液ADP转化为ATP),2mM鱼藤酮,5mM ADP和2mM琥珀酸盐作为呼吸底物(复合物II)。可以使用其他呼吸底物。
    7. 对每个样品测定使用补充有30μMOL的反应缓冲液的平行反应。该反应表明合成的ATP可能独立于H sup + +/-ATP合酶活性而发生。
    8. ATP合成反应的触发通过向含有450μl反应缓冲液(如步骤A6和A7所述)的管中加入150-200μg透化细胞或50μg分离的线粒体的蛋白质来实现在30℃下在恒定摇动下摇动管。
    9. 每30秒取少量等分试样(50μl),直至3分钟(反应终止),并加入含有200μl6%HClO 4的试管中。
    10. 将管立即涡旋并置于4℃下1小时以沉淀蛋白质
    11. 然后,将管在11,000×g离心5分钟,将所得上清液转移到另一个管中,在2μlpH指示剂的存在下用10%KOH(〜130μl)中和。
    12. 使用ATP生物发光测定试剂盒CLS II在具有光度计读板器的96孔板中测定每个样品中ATP的含量。为此,在每个孔中混合50μl中和的样品与50μl包含在试剂盒中并含有荧光素酶的反应缓冲液。在同一板中产生并加工ATP标准曲线(0-10μM),加入50μlATP标准品和50μl试剂盒中包括的反应缓冲液。
    13. ATP合成的初始速率通过估计每个样品中产生的ATP的pmol来确定。 ATP的ATP合成活性表示为nmoles of ATP/min/mg蛋白质(如代表性数据中所示,图1)。

  2. 动力学测定ATP合成活性
    1. 为了以动力学模式测定线粒体ATP合成活性,我们遵循(Vives-Bauza等人,2007)详述的方案,进行一些修改。
    2. 根据先前的协议进行步骤B1-4
    3. 将渗透的细胞或50-150μg的分离的线粒体重悬于40μlRM中。 将管保持在冰上直到使用。
    4. 在RM(参见配方2和3)中制备两种混合物A和B与反应化合物
    5. 在每个孔中加入160μl的混合物A和20μl的混合物B. 光度计读板器设置为在〜10分钟内每10秒以动态模式测量发光,其中发光线性地增加〜250秒。
    6. 将板放置在光度计,并添加20微升透化细胞或步骤B3中获得的分离的线粒体开始ATP合成的反应。
    7. 使用ATP标准曲线将相对光单位转化为ATP浓度。 为此,在混合物A中制备0和10μM之间的ATP溶液三份,终体积为180μl。 最后,向每个孔中加入20μl混合物B并测量终点的发光(参见图2)。
    8. ATP合成的初始速率通过估计每个样品中产生的ATP的pmol来确定。 酶的ATP合成活性表示为ATP/min/mg蛋白质的pmoles(如代表性数据中所示,图2)。

代表数据

  1. 为了将获得的RLU转化为ATP,有必要遵循以下步骤:
    1. 从不存在OL(T0,T1,T2,T3)时采取的时间点减去试管中的T0,T1,T2和T3时间点中的RLU + OL(由于线粒体中含有的ATP, (图1A)。在不存在OL的情况下(图1A),验证RLU线性地和显着地增加是非常重要的,而在OL的存在下RLU发生细微的或可忽略的变化(图1A)。
    2. 在线性回归(图1B)中插值获得的RLU值(图1A),以获得已测量的样品体积中的ATP含量。
    3. 将所获得的pmol的ATP乘以固定值7.6,其是总体积与测量的样品体积之间的比率。 (总体积=250μl+130μl=380μl); (样品体积=50μl); (比率= 7.6)(来自步骤A9和A11)。
    4. 将在点3下获得的值乘以指示在每个时间点取得的样品的体积的因子,相对于在T1处的反应总体积(即,,因子,因为450/50 = 9 ;因子8在T2,因为400/50 = 8;因子7在T3,因为350/50 = 7,...)(来自步骤A9)。
    5. 归一化由蛋白质量和反应时间获得的ATP的总pmol(图1C)
    6. 当使用透化细胞制剂的活性时,遵循相同的计算程序

      图1.在寡聚霉素(OL)不存在(-OL)或存在(+ OL)时来自小鼠心脏的分离的线粒体中的ATP合酶活性(终点模式)。A.相对光单位(RLU )的样品在0,1,2和3分钟后,在不存在和存在OL的情况下触发反应。 B. ATP量和荧光素/荧光素酶依赖性发光之间的相关性的线性回归分析。 C.使用三种心脏线粒体制剂的ATP合成活性(平均值±S.E.M)的实施例
  2. 为了将获得的RLU转化为ATP,必须遵循以下步骤:
    1. 从T0减去所有其他时间点的RLU(图2A)。
    2. 为了将反应的RLU转化为ATP量,需要在线性回归中在60秒内插RLU(图2B)。
    3. 根据样品中的蛋白质量,归一化数据是重要的(图2C)。
      注意:如果您使用RLU 60秒来内插到图2B,您的数据已经是一个速率(pmol ATP/min)。
    4. 当使用分离的线粒体制剂的活性时,遵循相同的计算程序

      图2.在没有(蓝色)或存在寡霉素(粉红色)的情况下在毛地黄皂苷透性化的HCT116细胞中的ATP合酶活性(动力学模式)。A.以相对光单位产生ATP的动力学表示(RLU)。灰色虚线表示ADP的磷酸化反应的初始速率,其为线性的至少60秒,并且其可以从线的斜率计算。 B. ATP量和荧光素/荧光素酶依赖性发光之间的相关性的线性回归分析。 C.使用三种制剂的洋地黄皂苷透性HCT116细胞的ATP合成活性(平均值±S.E.M.)的实施例。

笔记

  1. 毛地黄皂苷的浓度和透化时间是关键的,并且可能根据细胞系而改变。因此,我们建议先前滴定在每个细胞中使用的洋地黄皂苷浓度 线更适合ATP合成活性的测定。
  2. 建议在使用前新配制ADP

食谱

  1. 呼吸中(RM)(100 ml)
    225mM蔗糖(MW = 342.2965g/mol):7.7g/dm 2 10mM KCl(MW = 74.5g/mol):74mg
    5mM MgCl 2(MW = 95.2g/mol):47.6mg
    0.05%w/v牛血清白蛋白:50mg
    10mM磷酸钾缓冲液[HK 2 PO 4 4(MW = 174.17g/mol):0.17g; H sub 2 KPO 4(MW = 136.1g/mol):0.16g]
    1mM EGTA(MW = 380.35g/mol):38mg
    10mM Tris-HCl(pH7.4)(MW = 121.14g/mol):38mg
  2. 混合A
    呼吸中( 0.1 mM ADP
    5mM琥珀酸盐 0.15μMP 1,P - 二(腺苷-5')五磷酸盐
    2μg/ml鱼藤酮与或不与30μM寡霉素一起培养
  3. 混合B
    RM
    0.25mg/ml荧光素
    0.02mg/ml荧光素酶。

第三部分。 测定H + -ATP合酶的ATP水解活性

材料和试剂

  1. 微孔板,96孔,聚苯乙烯,黑色平底,TC处理(Cultek,目录号:153603)
  2. 蛋白质测定染料试剂浓缩物(Bio-Rad Laboratories,目录号:5000006)
  3. 液氮
  4. 磷酸酶抑制剂混合物2(Sigma-Aldrich,目录号:P5726)
  5. (Sigma-Aldrich,Roche Diagnostics ,目录号:11836170001)的微量培养基
  6. 寡霉素(VWR International,目录号:80058-538)
  7. (Sigma-Aldrich,目录号:T1503)
  8. 牛血清白蛋白(Sigma-Aldrich,目录号:A7906)
  9. 腺苷5'-三磷酸(ATP)二钠盐水合物(Sigma-Aldrich,目录号:FLAAS)
  10. KCl(Merck Millipore Corporation,目录号:104936)
  11. MgCl 2(Merck Millipore Corporation,目录号:172571)
  12. 羰基氰4-(三氟甲氧基)苯腙(FCCP)(Sigma-Aldrich,目录号:C2920)
  13. 抗霉素A(Sigma-Aldrich,目录号:A8674)
  14. 磷酸(烯醇)丙酮酸环己基铵盐(Sigma-Aldrich,目录号:P3637)
  15. 来自兔肌肉的L-乳酸脱氢酶(L-LDH)(Sigma-Aldrich,Roche Diagnostics ,目录号:10127876001)
  16. 来自兔肌肉的丙酮酸激酶(Sigma-Aldrich,目录号:P9136)
  17. β-烟酰胺腺嘌呤二核苷酸,还原二钾盐(NADH)(Sigma-Aldrich,目录号:N4505)
  18. 反应缓冲液(RB)储备溶液(见配方)
  19. FCCP储备溶液(见配方)
  20. 抗霉素A储备溶液(见配方)
  21. 完全反应缓冲液(见配方)
    1. 10mM PEP储液
    2. 2.5mM ATP储液
    3. 1mM NADH储备液
    4. 低聚霉素A储备溶液

设备

  1. FLUOstar Omega微孔板读数器(BMG Labtech,型号:FLUOstar Omega)
  2. Heraeus Megafuge 11R(Thermo Fisher Scientific,型号:Heraeus Megafuge 11R)
  3. 离心机5415R(Sigma-Aldrich,Eppendorf ,型号:5415R)

程序

  1. 可以在340nm处的吸光度变化(A 340)后,通过分光光度法测定酶对ATP的水解。该方法的基础是乳酸脱氢酶(LDH)和丙酮酸激酶(PK)催化的酶反应与ATP合酶的水解活性的偶联(Barrientos等人,2009)。通过ATP水解产生的ADP允许PK将1摩尔磷酸烯醇丙酮酸(PEP)转化为1摩尔丙酮酸。然后丙酮酸被LDH用于在该过程中产生乳酸盐将NADH分子氧化为NAD +。通过每个ATP分子水解,一分子NADH被氧化,允许通过A 340中的减少检测反应。
  2. 通过在液氮中和在37℃下分别冷冻和解冻线粒体制剂的三个循环,在分离的断裂的线粒体上测定H sup +/-ATP合酶的水解活性。
  3. 将30-50μg分离的线粒体重悬于20μl含有1x磷酸酶和蛋白酶抑制剂的反应缓冲液(RB)中。
  4. 设置发光计平板读数器以在动力学模式(每孔10次闪烁)中测量340nm处的吸光度,并且通过恒定的时间间隔(15秒)测量吸光度的变化。在这些条件下,可以同时测量24个样品。
  5. 将80μl完全RB(含PK和LDH)和20μl如步骤3制备的线粒体加入每个96孔黑色透明底板中,记录反应。
  6. 当产生足够的ATP水解时(2-5分钟),向每个孔中加入30μM寡霉素。加入寡霉素抑制线粒体ATP合酶/水解酶的活性,提供测定特异性的确认。
  7. 分离的线粒体的ATP酶活性以NADH氧化的纳米摩尔/min/mg线粒体蛋白表示,其使用Lambert-Beer方程计算并且NADH的摩尔消光系数为6.22×10 3 M < sup> -1 cm -1
  8. N =ΔA340 /(ε340×l)其中N是被氧化的NADH的浓度,ΔA340是两个给定时间点之间的吸光度的减量,ε340是摩尔浓度 NADH在340nm处的消光系数和l是1cm的光程长度

代表数据



图3.来自小鼠心脏的分离的线粒体中的ATP水解酶活性。图的斜率表示A 340作为反应时间的函数的递减。在指示的情况下,加入30μM寡霉素(+ OL,箭头)。分析含有60μg(A,红色)和30μg(B,蓝色)分离的线粒体的两种不同制剂。加入OL(封闭条)通过抑制反应的斜率阻断水解酶活性作为证据。 ATP水解酶活性表示为mU/mg蛋白。条代表平均值±S.E.M。的在来自小鼠心脏的分离的线粒体的三种制剂中测定的ATP酶活性。

笔记

  1. 注意,在步骤3添加的RB不补充PK和LDH
  2. ATP和NADH必须新鲜制备。
  3. FCCP,抗霉素A,PEP和寡霉素储备溶液可以在-20℃储存。

食谱

  1. 反应缓冲液(RB)储备溶液(10ml) 50mM Tris-HCl(pH8.0)(MW = 121.14g/mol):61mg
    50mM KCl(MW = 74.55g/mol):37.2mg
  2. 2.5 mM FCCP储液
    FCCP(MW = 254.17g/mol)
    将0.6mg FCCP溶于1ml DMSO中
  3. 500μM抗霉素A储备液
    抗霉素A(MW = 548.63g/mol) 将0.3mg抗霉素A溶于1ml乙醇中
  4. 完全反应缓冲液,1ml(足够体积的RB运行十次测定)
    1. 0.92ml RB储备液
    2. 2μl2.5mM FCCP原液(终浓度:5μM)
    3. 2μl500μM抗霉素A原液(终浓度:1μM)
    4. 10μl1M PEP(最终浓度:10μM)
      将27mg PEP溶解在100μl水中
    5. 25μl100mM ATP(终浓度:2.5mM) 将5.5mg溶于100μl水中
    6. 10μl100mM NADH(终浓度:1mM) 将7.4mg溶解在100μl水中
      13μl4单位LDH(2,750U/ml)
      20μl4单位PK(1,920U/ml)
      注意:在最后一步中和在通过加入线粒体触发反应之前进行4单位的LDH和4单位的PK的添加。
    7. 2μl1.5mM寡霉素A储液
      将1.2mg低聚霉素储备溶液溶解在1ml乙醇中

致谢

我们感谢博士。 MaríaSánchez-Aragó和Laura Formentini为设置ATP合酶活性的测定提供专家指导。认识到M. Chamorro和C.Nuñezde Arenas的技术援助。作者感谢Drs的实验室开发的以前的工作。 Enríquez,Barrientos和Manfredi,用于开发适用于这些方案的方法。 JGB和CNT得到了来自FPI-MICINN/MINECO和Fondo Social Europeo,Spain的博士后奖学金的支持。这项工作得到西班牙经济竞争部长(SAF2013-41945-R),马德里商会(S2011/BMD-2402)和拉美区域基金(FRA)的资助。 CBMSO从FRA获得机构资助。

参考文献

  1. Barrientos,A.,Fontanesi,F。和Diaz,F。(2009)。  使用极谱法和分光光度酶测定法评价线粒体呼吸链和氧化磷酸化系统。 19:Unit1913。
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引用:García-Bermúdez, J., Nuevo-Tapioles, C. and Cuezva, J. M. (2016). Determination of the H+-ATP Synthase and Hydrolytic Activities. Bio-protocol 6(16): e1905. DOI: 10.21769/BioProtoc.1905.
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