Isolation of Mitochondria from
Mammalian Tissues and Cultured Cells
Isolation of mitochondria can be a necessary procedure
for many purposes: (1) as a primary step for
further purification of mitochondrial subcomponents;
(2) to perform metabolic assays, with respiration activity
analysis the most common; and (3) to perform
molecular analyses on the biogenetic activity of the
organelle. The mitochondrial preparations obtained
using the method described herein are perfectly suitable
for biogenetical studies (mitochondrial DNA,
RNA, protein synthesis, and as source for respiratory
complex analysis by blue-native gel electrophoresis),
as well as in a variety of different assays where isolated
mitochondria are required, such as assessment of
respiratory enzyme activities, protein import, aminoacylation,
and in organello
footprinting. The method
consists of three basic steps: (1) cell rupture, (2) differential
centrifugation: first at low speed to pellet mainly
nuclei and unbroken cells and then at high speed to
pellet mitochondria, and (3) washing of the mitochondrial
pellet in order to reduce the presence of other
subcellular contaminants. Mitochondria from different
sources are obtained using basically the same methodology,
although some modifications must be introduced
depending on the tissue type or if mitochondria
are to be isolated from cultured cells. When the purity
of samples is considered, there is an elimination of a
good part of the contaminants when mitochondrial
preparations obtained by this procedure are compared
with crude mitochondrial fractions. Because this
purification protocol does not make use of gradient
preparation or ultracentrifugation, further purification
of the organelles is recommended when mitochondria
are prepared for isolation of organelle subcomponents.
In summary, the method described here produces reasonably
pure mitochondria in a fairly short time and
with a low cost. In addition, maintenance of integrity
and functionality of the organelles are guaranteed.
II. MATERIALS AND
Sucrose (ACS for analysis) is from Carlo Erba
Reagenti (Cat. No. 477183). Sodium chloride (ACS
analytical reagent, Cat. No. 727 810) is from Prolabo.
Potassium cyanide (KCN; BioChemika MicroSelect;
Cat No. 60178) is from Fluka. Ethylenediaminetetraacetic
acid disodium salt 2-hydrate (EDTA-Na2
analysis-ACS; Cat. No. 131669), potassium chloride
(for analysis-ACS-ISO; Cat. No. 131494), magnesium
chloride 6-hydrate (for analysis-ACS-ISO; Cat. No.
131396), sulphuric acid (96%; Cat. No. 251058),
trichloroacetic acid [solution 20% (w/v); Cat. No.
252373], acetic acid glacial (chemically pure; Cat. No.
211008), sodium hydrogen sulphite [solution 40%
(w/v); Cat. No. 211642], sodium sulphite (anhydrous
purissimum; Cat. No. 141717); dipotassium hydrogen
phosphate (anhydrous for analysis; Cat. No. 121512),
potassium dihydrogen phosphate (for analysis, Cat.
No. 121509) and orthophosphoric acid (85% for
analysis-ACS-ISO; Cat. No. 131032) are from Panreac.
D-Mannitol (ACS reagent; Cat. No. M-9647), ethylene
acid (EGTA; approximately 97%; Cat. No. E-4378), Lglutamic
acid monosodium salt [glutamate; minimum
99% (TLC); Cat. No. G-1626], L
-(-)-malic acid sodium
salt [malate; 95-100% (enzymatic); Cat. No. M-1125],
succinic acid disodium salt hexahydrate (succinate; minimum 99%; Cat. No. S-2378), oxalacetic acid
(approximately 98%; Cat. No. O-4126), acetyl coenzyme
A trilithium salt (acetyl-CoA; approximately
95%, Cat. No. A-2181), 5-5'-ditio-bis(2-nitrobenzoic
acid) or DTNB (Ellman's reagent; Cat. No. D-8130),
glycerol 2-phosphate disodium salt hydrate (β-
glycerophosphate; ≤ 0.1% α-isomer; Cat. No. G-6251),
ammonium molybdate tetrahydrate (81-83% as MoO3
ACS reagent; Cat. No. A-7302), β-D
phosphate sodium salt (crystalline, Sigma Grade; G-
7879), imidazole [minimum 99% (titration), crystalline;
Cat. No. 1-0250], hydrogen peroxide (29-32% as H2
ACS reagent; Cat. No. H-0904), bovine serum albumin
[BSA; fraction V; minimum 96% (electrophoresis); Cat.
No. A-4503], and subtilisin (subtilisin A from Bacillus
sp.; lyophilized powder, type VIII, 7-15 units/mg
solid; Cat. No. P-5380) are from Sigma. Aldrich supplied
the titanium (IV) oxysulfate [titanyl sulfate or
; 15% (w/v) solution in diluted H2
; Cat. No.
49,537-9] and the 4-amino-3-hydroxy-1-naphtelenesulphonic
acid (aminonaphtosulphonic Acid; 98+% ACS
reagent; Cat. No. 39,896-9). D
-Sorbitol (high purity;
Cat. No. SO0850) is from Scharlau. Tris (for analysis;
Cat. No. 1.08382), hydrochloric acid fuming (37%; Cat.
No. 1.00317) and 2-mercaptoethanol (for synthesis;
Cat. No. 8.05740) are from Merck. BSA fraction V,
fatty acid free (Cat. No. 775 827), adenosine-5'-
diphosphate disodium salt (ADP; Cat. No. 127 507),
cytochrome c (from horse heart, salt free, Cat. No. 103
888), and Triton X-100 (especially purified for membrane
research; Cat. No. 789 704) are from Roche. For
protein determination, the Bio-Rad protein assay dye
reagent concentrate (450ml; Cat. No. 500-0006) is
used. Phosphate-buffered saline (PBS 1×, liquid, pH
7.4 ± 0.05; Cat. No. 10010) is from GIBCO (Invitrogen
Elvejhem-type glass homogenisers with Teflon
pestles, as well as the Dounce-type glass potter, are
from local glassware providers (VidraFoc and
Sumalsa). Fifty-milliliter polypropylene copolymer
centrifuge tubes (Oak Ridge centrifuge tubes; Cat. No.
3119-0050) and the 0.2-µm syringe filters (Cat. No.
190-2520) are from Nalgene.
Centrifugations are performed in a Sorvall RC 5B
Plus refrigerated centrifuge with the Sorvall HS-4
swinging rotor and the SS-34 fixed angle rotor. An
Eppendorf 5415 C microfuge kept in the cold room is
used to centrifuge the Eppendorf tubes.
Mitochondrial oxygen consumption measurements
are obtained with a Hansatech CBID oxygen electrode
and registered in a PC using software from Pico Technology
Limited. The polytetrafluoroethylene (PTFE)
membrane (ordering code: S4; thickness 0.0125mm,
width 25.4mm, 30m reel) is from Hansatech.
Spectrophotometric measurements are performed
in a Unicam UV 500 spectrophotometer, where the
temperature is kept constant by a DBS PCD 150 water
peltier system. Data from these measurements are
registered by a PC using the Vision 32 version 1.25
software from Unicam Limited.
A. Isolation of Mitochondria
2. Isolation of Mitochondria from Rat Tissues
a. Mitochondria from Liver and Kidney
- Homogenisation medium A: 0.32M sucrose, 1mM EDTA, and 10 mM Tris-HCl. For 1 liter, weigh 109.5 g
of sucrose, dissolve in bidistilled water, and add 2ml
of 0.5M disodium EDTA stock solution and 20ml of
0.5 M Tris-HCl, pH 7.4. Check and adjust, if necessary,
pH to 7.4 and autoclave at low pressure. Store at 4°C.
- Homogenisation medium AT: 0.075M sucrose,
0.225M mannitol, 1mM EGTA, and 0.01% BSA. For
500ml, add 12.84g of sucrose, 20.5g of mannitol, and
5 ml of a stock solution of 100 mM EGTA. Adjust pH to
7.4 and autoclave at low pressure. Store at 4°C. Just
before use add 500µl of 10% (w/v) BSA. Once the BSA
is added, the solution cannot be autoclaved again.
- Homogenisation medium IB 10×: 0.35M Tris-HCl,
pH 7.8, 0.25M NaCl, and 50mM MgCl2. To prepare
100 ml, add 70 ml of 0.5 M Tris-HCl, pH 7.8, 5 ml of 5 M NaCl, and 5 ml of 1M MgCl2. Complete to 100 ml with
bidistilled water and sterilize by filtration. Store at 4°C.
- MAITE medium: 25 mM sucrose, 75 mM sorbitol,
100 mM KCl, 0.05 mM EDTA, 5 mM MgCl2, 10 mM Tris-HCl, pH 7.4, and 10mM H3PO4 (pH 7.4). For
100 ml of solution weigh 0.86 g of sucrose, 1.37 g of sorbitol,
and 0.74 g of KCl, dissolve, and then add 10 µl of
0.5 M EDTA, 500 µl of 1M MgCl2, 2 ml 0.5 M Tris-HCl,
pH 7.4, and 68 µl of orthophosphoric acid. Adjust pH to
7.4 using Tris base 0.5 M. Sterilize by filtration through a
0.2-µm filter, make 10-ml aliquots, and store at 4°C.
- 0.5M EDTA: Weigh 93.1 g of disodium EDTA,
dissolve, adjust the pH to 8.0, and complete the
volume to 500ml with distilled water. Store at 4°C.
- 0.5 M Tris-HCl pH 7.4 or pH 7.8: For 500 ml, weigh
30.3 g of Tris and adjust pH to 7.4 or 7.8 with HCl. Store
- 100 mM EGTA: Prepare 100 ml of this solution by
weighing 3.8g. In order to increase the solubility of
EGTA, the pH has to be raised with NaOH when dissolving
it. Once the EGTA is totally dissolved, complete
to 100ml with bidistilled water. Store at 4°C.
- 1M MgCl2: For 100ml, weigh 2 g of magnesium
chloride and dissolve. Store at 4°C.
- 5M NaCl: Weigh 29.3g of NaCl, dissolve, and
adjust to 100 ml with bidistilled water.
- 10% (w/v) or 100mg/ml BSA: Weigh 0.5g of fatty
acid-free BSA and dissolve in 5 ml of distilled water.
Sterilize by filtration through a 0.2-µm filter and store
at -20°C in 1-ml aliquots.
Use male Wistar rats weighing 200-300g. It is recommended
that glassware, scissors, and metal sieves
be sterilized at 160°C overnight. Autoclave plastic
tubes at 1 atm for 20min.
- Previous to the sacrifice of the animals, fill
100-ml glass beakers with enough homogenisation
medium A to cover the organ to be utilised, weigh, and
place them on ice.
- Kill the animals, remove the organ of interest,
and place it in a beaker with ice-cold homogenisation
medium A (in this case liver or kidneys).
- Weigh the beaker to know the amount of tissue
that has been extracted.
- Cut the tissue into small pieces with a pair of
- Sift the tissues and wash them with more
homogenisation medium in order to remove blood and
connective tissue. Put them back in the beaker and
repeat steps 4 and 5 three or four times until the tissue
is well cleaned.
- Add fresh homogenisation medium to the
homogeniser in a proportion of 4 ml per gram of liver
and 5 ml per gram of kidney.
- Transfer the pieces of tissue to the homogeniser.
- Homogenise with four up-and-down strokes in
the Elvehjem-Potter with the motor-driven Teflon
pestle at 600rpm.
- Transfer the homogenised tissue into the 50-ml
sterile centrifuge tubes.
- Centrifuge in the HS-4 swinging rotor at 1000g (3000rpm) for 5 min at 4°C in order to pellet unbroken
tissue, cells, and nuclei.
- Place eight Eppendorf tubes in a container with
ice and fill them with supernatant from the previous
step (this and the following steps are done in a cold
- Centrifuge the Eppendorf tubes for 2min at
13,000rpm in a microfuge.
- Using a sterile Pasteur pipette, remove the
supernatant trying to draw out all the liquid and
part of the light coloured fluffy layer on the top of
the pellet without disturbing the darker part where
- To wash the mitochondrial fraction, add
homogenisation medium A, up to 1.5 ml, to four of the
eight Eppendorf tubes and resuspend. Transfer the
resuspended pellet of one tube to one of the tubes
without medium A and resuspend.
- Centrifuge the four resulting tubes as described
- Repeat steps 15, 16, and 17 so that now two
tubes are centrifuged.
- Repeat the same procedure once again until one
tube, containing all the material, is left.
- When the last tube is centrifuged and the supernatant
is removed, wash the pellet by resuspending it
with MAITE medium.
- Centrifuge for 2min at 13,000rpm.
- Resuspend the pellets with 1 ml of MAITE
When a bigger amount of sample is needed, the procedure
can be started using twice as much supernatant
and then finishing with the mitochondrial preparation
in two Eppendorf tubes.
b. Mitochondria from Heart
- Follow the steps 1 to 7 described in Section
III,A,2,a but instead of filling the beakers with homogenisation
medium A, use homogenisation medium AT.
- Fill the homogeniser with 10ml of medium AT
per gram of heart.
- Homogenise with six strokes in the Elvehjem-
Potter with the motor-driven Teflon pestle at 600rpm.
- Transfer the homogenised tissue to a sterile
50-ml centrifuge tube.
- Centrifuge at 1000g (3000rpm) in the HS-4
swinging rotor for 5 min at 4°C.
- Transfer the supernatant from the previous centrifugation
step to a clean centrifuge tube.
- Centrifuge at 12,000g (9000rpm) in the SS-34
fixed angle rotor for 10min at 4°C to obtain the crude
- Draw out and discard the supernatant.
- Resuspend the pellet in 5.5 ml of homogenisation
- Transfer the mitochondrial suspension to four
Eppendorf tubes for the washing steps in the cold
- Proceed hereafter as described for liver and
kidney mitochondria, but if the mitochondrial fraction
is obtained from one rat heart, the final fraction has to
be resuspended in a smaller volume than those from
liver and kidney, usually with 0.5-0.75 ml.
An alternative isolation procedure for heart mitochondria
has been described previously (McKee et al.
1990) where perfusion and homogenisation of the hearts using subtilisin (0.4mg/ml) are proposed to
improve the yield, the respiratory performance, and
the protein synthesis activity of the isolated organelles.
In our hands, the addition of subtilisin to the
homogenisation medium provides a higher yield in
the preparations and increases the in organello
rate. However, no significant improvement in
the rate of incorporation of a radioactive amino acid
into the mitochondrial translation products could be
observed. However, the electrophoretic pattern of in
organello synthesized products reveals an abnormal
accumulation of low molecular weight peptides
when subtilisin is used, probably due to residual
peptidase activity of the subtilisin after breaking the
c. Mitochondria from Brain
- Again follow the same first five steps as for liver,
kidney, and heart. Use homogenisation medium AT.
- Add 5 ml of medium AT per gram of brain to the
- Homogenise the pieces of brain with 10 to 15
strokes using a Dounce-type glass homogeniser with a
manually driven glass pestle.
- Transfer the homogenised tissue to a sterile
50-ml centrifuge tube.
- Centrifuge at 1000g in swinging rotor for 5 min
- Transfer the supernatant from the previous centrifugation
step to a clean sterile centrifuge tube.
- Resuspend the pellet resulting from the previous
centrifugation in another 5 ml of medium AT per gram
of starting tissue.
- Rehomogenise the nuclear pellet by repeating 10
to 15 strokes in the Dounce potter.
- Transfer the homogenised nuclear pellet to the
same 50-ml tube where the first centrifugation was
- Centrifuge at 1000g in the swinging rotor for
5 min at 4°C.
- Remove this second supernatant and pour it
into the tube with the first supernatant.
- Centrifuge at 12,000g (9000rpm) in the fixed
angle SS-34 rotor for 10min at 4°C to obtain the crude
- Wash and resuspend as described for heart
mitochondria (steps 8-11).
- Resuspended the final pellet in 0.9 ml of MAITE
A great fraction of mitochondria is lost in the first
nuclear pellet in whatever tissue is used; therefore,
these steps (7-11) of rehomogenisation and a second
centrifugation are especially necessary when isolating brain mitochondria because the homogenisation is
much gentler than for the other tissues and the yield
is too low when only one homogenisation step is done.
The brain mitochondrial fraction obtained this way
contains free as well as synaptic mitochondria. To separate
free mitochondria from synaptosomes ("pinched
off" synaptic ends with the synaptic mitochondria),
several methods are available (reviewed in Whittaker,
1993). Partitioning in an aqueous two-phase system is
recommended (Lopez-Perez, 1994).
3. Isolation of Mitochondria from Mammalian
This procedure is the modified Gaines method
(Enriquez and Attardi, 1996; Fernandez-Vizarra et al.
2002; Gaines, 1996).
B. Assessment of Purity
- Harvest exponentially growing cells by
trypsinization. Use the cells contained in ten to twelve
150-mm plates that are about 80-90% confluent.
- Transfer the cells to a 50-ml Falcon tube.
- Wash twice with cold PBS by centrifuging at
600g in the clinical centrifuge for 8 min at 4°C. In the
last wash, transfer the cells to a 15-ml Falcon tube and
centrifuge as before.
- Place the cells in ice. Measure the volume occupied
by the packed cells.
- Resuspend the cells in one-half of the packed
cell volume of IB 0.1×. This is a hypotonic medium to
facilitate the breakage of the cells.
- Pipette the cell suspension into the homogeniser.
- Wash the tube where the cells are with 0.1× IB
using another half volume of the originally packed
cells and pipette this into the homogeniser.
- Perform four to five strokes in the homogeniser
with the motor-driven Teflon pestle at 600rpm.
- Immediately add 1/10 of the initial volume of
packed cells of 10× IB, to make the medium isotonic.
- Transfer the homogenised cells to a 15-ml
- Centrifuge at 1600g (3500rpm) for 3min at 4°C in the HS-4 swinging rotor to pellet unbroken cells,
debris, and nuclei.
- Draw out the supernatant and transfer it to a
clean 15-ml Falcon tube.
- Resuspend the nuclear pellet in one-half of its
volume of 0.1× IB.
- Repeat steps 6 to 11.
- Collect the supernatant from this second round
of homogenisation and centrifugation and add it to the
- Centrifuge the supernatants at 1600 g (3500 rpm)
for 3 min at 4°C in the HS-4 swinging rotor to remove
remaining nuclei and unbroken cells.
- Pipette the supernatant in (normally two)
Eppendorf tubes placed in ice (this one and the following
steps are performed in the cold room).
- Centrifuge the Eppendorf tubes at 13,000rpm in
the microfuge for 1 min.
- Wash the mitochondrial pellet with homogenisation
medium 1× IB, until all the material is in one tube.
- Remove supernatant and wash the pellet by
resuspending it with homogenisation medium A.
- Centrifuge the Eppendorf tube at 13,000rpm in
the microfuge for 1 min.
- Remove supernatant and wash the pellet using
- Centrifuge the Eppendorf tubes at 13,000rpm in
the microfuge for 1 min.
- Resuspend the mitochondrial pellet in the
appropriate volume of MAITE medium, which is
usually around 300 µl.
Two different parameters have to be considered
when assessing the purity of a mitochondrial fraction.
One is the enrichment of the preparation in mitochondria
and the other is the presence of contaminants. The
enrichment in mitochondria is evaluated by measuring
the activities of mitochondrial enzymes in the
initial homogenate and in the final mitochondrial
preparation. We usually measure two activities: the
inner membrane-bound respiratory complex IV or
cytochrome c oxidase (Wharton, 1967) and the mitochondrial
matrix enzyme citrate synthase (Srere, 1969).
Spectrophotometric measurement of individual respiratory
complex activities has been reviewed previously
(Birch-Machin and Turnbull, 2001; Trounce,
1996). However, to evaluate the presence and abundance
of contaminants, different approaches can be
proposed. The most common contaminants in a mitochondrial
preparation are microsomes (mostly derived
from endoplasmic reticulum), lysosomes, and peroxisomes,
and their presence is monitored by the determination
of specific enzyme activities present in each
contaminant particle. Good examples of these activities
that we have used to assess purity are glucose-6-
phosphatase for endoplasmic reticulum (Morr6, 1971),
acid phosphatase for lysosomes (Trouet, 1974), and
catalase for peroxisomes (Baudhuin, 1974). Electron
microscopy morphometric analysis is good for estimating
unidentified contaminants for which no enzymatic
marker is available (Enriquez et al.
1. Treatment of Samples
Total homogenate samples that are used for spectrophotometric
enzymatic activity measurements must undergo a freeze-thawing treatment in order to break
the cells completely and liberate the enzymes from
the subcellular particles. Crude mitochondrial fractions
and mitochondrial preparations do not need such
treatment, they are just divided in aliquots and kept at
-70°C until they are used for the spectroscopic measurements.
The single freezing and thawing step is
sufficient to break them.
2. Cytochrome c Oxidase Activity (EC 22.214.171.124)
- Use 1-ml aliquots of total homogenate that are in
- Prepare a water bath at a temperature of 37°C.
- Fill an appropriate container with liquid nitrogen.
- Put the samples in the liquid nitrogen until they are
completely frozen (2 min).
- Immediately pass the samples to the water bath at
37°C and keep them there until they are thawed
completely (5 min).
- Repeat steps 4 and 5 four or five times.
- Aliquot the homogenate in Eppendorf tubes, 50-
100 µl in each tube, and keep them at -70°C.
Measurements of cytochrome c oxidase activity are
performed spectrophotometrically using 5 µl of sample
(nondiluted total homogenate or 1/10 diluted mitochondrial
samples) in a final volume of 1 ml. The
decrease of absorbance at 550nm, due to the oxidation
of cytochrome c, is measured for 90 s at 38°C (Wharton,
1967). Sensitivity to KCN is used to confirm that
cytochrome c oxidase activity is measured.
3. Citrate Synthase Activity (EC 126.96.36.199)
To measure citrate synthase activity, use the same
amount of sample as in the case of cytochrome c activity;
the final reaction volume is also 1 ml. In the spectrophotometer,
measure the increase of the absorbance
at 412nm due to the formation of a yellow complex
of free CoA with DTNB, for 90s at 30°C (Srere, 1969).
The CoA is formed in the reaction of acetyl-CoA
with oxalacetate to form citrate, catalysed by citrate
Mitochondrial enrichment assessed by these enzymatic
activities in the indicated preparations is shown
in Table I.
4. Glucose-6-phosphatase Activity (EC 188.8.131.52)
Glucose-6-phosphatase activity was measured
according to (Morré, 1971), determining spectrophotometrically
the inorganic phosphate (Pi
) liberated by the
enzyme from the substrate (glucose-6-phosphate). The
amount of sample used is 0.1ml, diluted 1/10 in
the case of total homogenate and 1/20 for mitochondria and crude mitochondrial fractions. After a 15-min
incubation at 37°C, there will be 0.1-1 µmol of Pi
milliliter. Protein is removed by TCA precipitation and
centrifugation, and the amount of inorganic phosphate
is measured in 1 ml of cleared supernatant.
5. Acid Phosphatase Activity (EC 184.108.40.206)
Incubate 1 ml of 1 / 1 0 diluted sample (total
homogenate, mitochondria, or crude mitochondrial
fractions) with 200 µl 0.5 M
β-glycerophosphate, 100 µl
of buffer, 100µl of 2% (w/v) Triton X-100, and 600µl
of water for 30min at 37°C (Trouet, 1974). Remove
protein by TCA precipitation and centrifugation and
measure the amount of inorganic phosphate in 1 ml of
The unit of activity is defined as the amount of
enzyme liberating 1 µmol of phosphate per minute.
6. Catalase Activity (EC 220.127.116.11)
Catalase activity in the samples is measured using
the method described in Baudhuin (1974), which is
based on the formation of the yellow titanyl sulphate-
complex. Liver and kidney samples must
be diluted (1/40 for liver and 1 / 1 0 for kidney),
whereas heart and brain samples do not need to be
diluted. After incubation for 10min at 0°C and addition
of the titanyl sulphate solution, measure the
absorbance at 405 nm to evaluate how much of the initially
is left. To calculate the activities, take
into account that the reaction follows first-order
kinetics and that one unit of enzyme is defined as the
amount consuming 90% of the H2
present in a 50-ml
reaction volume in 1 min.
Evaluation of the presence of contaminants is
shown in Table II.
C. Yield of Mitochondria and Normalisation
The yield of mitochondria depends on the source of
the organelles. Typically we obtain 6-9mg of mitochondrial
protein per gram of starting tissue for the
liver samples, which is 5-6mg/g for kidney, 2-3mg/g for heart, and 3-4.5mg of mitochondrial
protein per gram of tissue in brain samples. The yield
can also be calculated by the amount of mitochondrial
activity (cytochrome c oxidase and citrate synthase)
recovered in preparations from the total homogenate.
Table I shows values obtained for the different mitochondrial
preparations. The yield of mitochondria
evaluated this way varies from 8% of recovery in brain
to about 25% in liver.
Classically, the way of normalising mitochondrial
parameters is using protein content in the sample.
However, mitochondrial protein content in the different
preparations is very variable. In each kind of
sample, the nature and amount of contaminants vary,
and even the protein composition of mitochondria is
different depending on their source. In this way, specific
mitochondrial enzyme activities are very different
among organs; this is due to their intrinsic differences
in activity and also to the different protein content in
each preparation (Table I). More recently, citrate synthase
activity is often taken to normalise mitochondrial
parameters (Trounce, 1996), particularly respiratory
chain enzyme activities, because it is considered a
measurement of mitochondrial volume. However,
the intertissue variation on citrate synthase specific
activities is also too high and its use for normalization
when comparing different sources of organelles
is again questionable; i.e., when calculating the
cytochrome c oxidase/citrate synthase ratio, there
are significant differences between organs, with
liver apparently the highest cytochrome c oxidase
activity (these can easily be calculated from Table I
Taking all this into account, we propose that the best
way to normalise mitochondrial parameters when
comparing organelles from different sources is to
extract mitochondrial DNA from the organelle preparations
and total cell DNA from the homogenate of the
same samples and quantify the amount of nuclear
DNA and mitochondrial DNA. The quantification of
DNA is performed easily by conventional slot blot or
Southern blot and hybridisation using a specific probe
for each genome (Diez-Sanchez et al.
, 2003). Routinely
we use 12S rRNA for mtDNA and 18S rRNA for nDNA
using the following rat polymerase chain reactiongenerated
- 12S rRNA 617-bp amplification product (12S1
forward: caccgcggtcatacgattaacc; 12S2 reverse:
- 18S rRNA 429-bp amplification product (18S1
forward: cctcgatgctcttagctgagtg: 18S2 reverse:
In this way, the parameter/nuclear DNA ratio
would represent a "per cell" or "per genome" estimation
and the parameter/mtDNA a "per mitochondria"
or "per mitochondrial DNA" estimation. Although the
mtDNA copy number per cell varies among cell types,
tissues, and physiological condition, it can be monitored
easily by following the mtDNA/nDNA ratio.
D. Assessment of Functionality
Mitochondria obtained using the purification protocols
described here have intact membranes and are
coupled. This means that electron transfer among the
inner membrane complexes takes place when there is
phosphorylation of ADP by the ATP synthetase; this is
the main way to dissipate the proton gradient between
the intermembrane space and the matrix. Mitochondrial
electron transfer can be measured by oxygen consumption
using an oxygen electrode (Trounce, 1996).
The respiratory control ratio (RCR) is a way to determine
how coupled mitochondria are The consumption
of oxygen by mitochondria in the presence of electron
donors (substrates) and ADP, called state 3 respiration,
is measured and then compared with the respiration
rate when all the ADP has been phosphorylated (state
4). When there is no difference between state 3 and
state 4 respiration (RCR = 1), mitochondria are completely
uncoupled, whereas RCRs higher than 4 are
indicative of tightly coupled mitochondria. Another
parameter to evaluate coupling using the same measurements
in the oxygen electrode is the P/O ratio,
which gives the moles of synthesized ATP per atom of
oxygen transformed to water during oxidative phosphorylation.
Coupled mitochondria usually exhibit
P/O ratios higher than 2.5, approaching 3 with NADlinked
substrates, and ratios higher than 1.8, approaching
2 with succinate (Palloti and Lenaz, 2001). A
detailed explanation on oxygen electrode functioning
can be found in Rickwood (1987).
- Perform measurements in the oxygen electrode
using MAITE medium where mitochondria are resuspended.
The final volume in the electrode chamber is
- Add 10µl of 100mg/ml BSA and either 20µl of
500mM glutamate and 25µl of 100mM malate when
measuring RCR with glutamate and malate (NAD-linked substrates) or, as an alternative, 20µl of 500 mM succinate.
- Add 0.5 to 1 mg of mitochondrial protein from
freshly isolated mitochondria. In the case of heart
mitochondria, 0.2mg of protein is enough to perform
- When the chamber is closed, add 100nmol of
ADP (10µl of ADP 10mM) to stimulate mitochondria
and start state 3 respiration. This addition is repeated
after mitochondria return to state 4 respiration.
Values for RCR and P/O for different mitochondrial
preparations are given in Table III.
IV. COMMENTS AND
We would like to make a particular comment on the
nature of the less commonly evaluated contaminants in
the mitochondrial preparations, whose presence is
variable depending on their source and that has been
frequently underscored in classical preparative or
metabolic assays. In addition to their utility in bioenergetic
and metabolic studies, isolated organelles provide
a unique tool to investigate the synthesis and expression
of mtDNA in conditions that very much resemble
the in vivo
enzyme/substrate proportions, ionic composition,
and integrated activity of the metabolic and biogenetic
processes (Enriquez et al.
, 1996, 1999; Enriquez
and Attardi, 1996; Fernandez-Vizarra et al.
In this type of assay the presence of contaminants
can be relevant or negligible depending on the type of investigations to be performed. For example, only
brain crude mitochondria preparations contain large
myelin membrane debris that can be estimated by
monitoring the 2', 3'-cyclic nucleotide 3'-phosphohydrolase
(Enriquez et al.
, 1990; Olafson et al.
, 1969). They
do not seem to interfere with biogenetic analyses, but
can contribute to the overall protein content (see later).
In addition, this crude preparation contains synaptosomes
(also enclosing mitochondria). Crude mitochondrial
preparations are suitable for in organello
transcription and replication analysis when labeled
UTP or dNTPs are used, as well as for protein import
assays, as nucleotides or peptides do not cross the
plasma membrane of synaptosomes. However, when
analyzing protein synthesis and amino acylation using
labeled amino acids, one should keep in mind that
amino acids are also imported by synaptosomes and
used by their mitochondria. Therefore, if trying to
evaluate the influence of specific factors in the mitochondrial
protein synthesis, one has to be aware that a
relevant portion of mitochondria are protected by a
Other relevant contaminants, not considered very
often, are residual but partially active biogenetic components
of the nucleocytoplasmic machinery, such as
cytoplasmic ribosomes, transcriptionally or replicative
partially active nuclear rests, or cytoplasmic aminoacyl
tRNA synthetases. The use of additional purification
steps using density gradient purification could
reduce the presence of some of these contaminants, but
it is expensive, not suitable for large-scale preparations,
and, most important, can affect the functionality
of the purified organelles. Protein synthesis due to contaminant
cytoplasmic ribosomes is not very relevant
(Fernandez-Vizarra et al.
, 2002) and it is possible to
eliminate it completely using drugs that specifically
inhibit cytoplasmic protein synthesis without affecting
mitoribosomes such as cycloheximide or emetine. In
our hands, no detectable aminoacylation of cytoplasmic
tRNAs is observed when using purified organelles
from cultured cells (Enriquez, 1996). The same is
true for transcription in any source of mitochondria
tested (Andreu et al.
, 1998; Enríquez, 1999; Enriquez et al.
, 1991, 1996; Fernandez-Vizarra et al.
, 2002; Micol et al.
, 1997), but a background of nonmitochondrial
DNA is labeled when mitochondria are prepared
from exponential growing cultured cells (unpublished
results). To avoid this, it is recommended to add
DNase (or microccocal nuclease) during the mtDNA
replication assay, as mtDNA and mtRNAs are
protected by the mitochondrial double membrane.
Then, as the standard DNA isolation procedure, after
incubations, includes a strong step of proteinase K
digestion, the DNase is fully removed before breaking of the mitochondria. In that way only mitochondrial
DNA is labeled.
- Always keep mitochondrial preparations on ice
while they are being isolated and until they are used
for biogenetical analyses or for measurements in the
- Invert the tubes where the mitochondria are kept
every once in a while in order to maintain mitochondria
in suspension, avoiding sedimentation. During
the incubation for activity determination, mixing is
essential, as sedimentation of the organelles can make
them become permanently impaired in their biogenetic
activities due to a lack of oxygenation.
- Irreversible uncoupling of mitochondria during
purification can occur and is found more often when
isolating brain or heart mitochondria. This is likely due
to the well-known uncoupling activity of fatty acids
and could be prevented by the inclusion of fatty acidfree
BSA in the purification buffer.
We thank Drs. Julio Montoya, Acisclo Pérez-Martos,
and Manuel, José López-Pérez for their valuable input
in our work and Santiago Morales for his technical
assistance. Our research was supported by the Spanish
Ministry of Education PM-99-0082 grant to JAE, by the
Ramón y Cajal 2001 grant to PF-S, and by a Diputación
General de Arag6n (CONSID B015/2001) fellowship to
Andreu, A. L., Arbos, M. A., Perez-Martos, A., Lopez-Perez, M. J.,
Asin, J., Lopez, N., Montoya, J., and Schwartz, S. (1998). Reduced
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