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 INSTRUMENTATION
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; for 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 glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic 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-glucose 6- phosphate sodium salt (crystalline, Sigma Grade; G- 7879), imidazole [minimum 99% (titration), crystalline; Cat. No. 1-0250], hydrogen peroxide (29-32% as H2O2 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 TiOSO4; 15% (w/v) solution in diluted H2SO4; 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 Corporation).
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
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.
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
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 transcription 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 organelles.
c. Mitochondria from Brain
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 Cultured Cells
This procedure is the modified Gaines method (Enriquez and Attardi, 1996; Fernandez-Vizarra et al., 2002; Gaines, 1996).
B. Assessment of Purity
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., 1990).
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 184.108.40.206)
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 220.127.116.11)
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 synthase.
Mitochondrial enrichment assessed by these enzymatic activities in the indicated preparations is shown in Table I.
4. Glucose-6-phosphatase Activity (EC 18.104.22.168)
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 per 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 22.214.171.124)
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 cleared supernatant.
The unit of activity is defined as the amount of enzyme liberating 1 µmol of phosphate per minute.
6. Catalase Activity (EC 126.96.36.199)
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- H2O2 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 added H2O2 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 H2O2 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 Criteria
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 values).
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 probes.
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).
Values for RCR and P/O for different mitochondrial preparations are given in Table III.
IV. COMMENTS AND RECOMMENDATIONS
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., 2002).
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 plasma membrane.
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.
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 EF-V.
Andreu, A. L., Arbos, M. A., Perez-Martos, A., Lopez-Perez, M. J., Asin, J., Lopez, N., Montoya, J., and Schwartz, S. (1998). Reduced mitochondrial DNA transcription in senescent rat heart. Biochem. Biophys. Res. Commun. 252, 577-581.
Baudhuin, P. (1974). Isolation of rat liver peroxisomes. Methods Enzymol. 31, 356-368.
Birch-Machin, M. A., and Turnbull, D. M. (2001). Assaying mitochondrial respiratory complex activity in mitochondria isolated from human cells and tissues. Methods Cell Biol. 65, 97-117.
Diez-Sanchez, C., Ruiz-Pesini, E., Lapena, A. C., Montoya, J., Perez- Martos, A., Enriquez, J. A., and Lopez-Perez, M. J. (2003). Mitochondrial DNA content of human spermatozoa. Biol. Reprod. 68, 180-185.
Enríquez, J. A., and Attardi, G. (1996). Analysis of Aminoacylation of Human Mitochondrial tRNAs. Methods Enzymol. 264, 183-196.
Enríquez, J. A., Fernandez-Silva, P., Garrido-P6rez, N., L6pez-P6rez, M. J., P6rez-Martos, A., and Montoya, J. (1999). Direct regulation of mitochondrial RNA synthesis by thyroid hormone. Mol. Cell Biol. 19, 657-670.
Enriquez, J. A., Lopez-Perez, M. J., and Montoya, J. (1991). Saturation of the processing of newly synthesized rRNA in isolated brain mitochondria. FEBS Lett. 280, 32-36.
Enriquez, J. A., Perez-Martos, A., Lopez-Perez, M. J., and Montoya, J. (1996). In organello RNA synthesis system from mammalian liver and brain. Methods Enzymol. 264, 50-57.
Enriquez, J. A., Sanchez-Prieto, J., Muino Blanco, M. T., Hernandez- Yago, J., and Lopez-Perez, M. J. (1990). Rat brain synaptosomes prepared by phase partition. J. Neurochem. 55, 1841-1849.
Fernandez-Vizarra, E., Lopez-Perez, M. J., and Enriquez, J. A. (2002). Isolation of biogenetically competent mitochondria from mammalian tissues and cultured cells. Methods 26, 292-297.
Gaines, G. L., 3rd (1996). In organello RNA synthesis system from HeLa cells. Methods Enzymol. 264, 43-49.
Lopez-Perez, M. J. (1994). Preparation of synaptosomes and mitochondria from mammalian brain. Methods Enzymol. 228, 403-411.
McKee, E. E., Grier, B. L., Thompson, G. S., and McCourt, J. D. (1990). Isolation and incubation conditions to study heart mitochondrial protein synthesis. Am. J. Physiol. 258, E492-E502.
Micol, V., Fernandez-Silva, P., and Attardi, G. (1997). Functional analysis of in vivo and in organello footprinting of HeLa cell mitochondrial DNA in relationship to ATP and ethidium bromide effects on transcription. J. Biol. Chem. 272, 18896-18904.
Morré, D. J. (1971). Isolation of Golgi apparatus. Methods Enzymol. 31, 130-148.
Olafson, R. W., Drummond, G. I., and Lee, J. F. (1969). Studies on 2',3'-cyclic nucleotide-3'-phosphohydrolase from brain. Can. J. Biochem. 47, 961-966.
Palloti, E, and Lenaz, G. (2001). Isolation and subfractionation of mitochondria from animal cells and tissue culture lines. Methods Cell Biol. 65, 1-35.
Rickwood, D., Wilson, M. T., and Darley-Usmar, V. M. (1987). Isolation and characteristics of intact mitochondria. In "Mitochondria: A Practical Approach" (V. M. Darley-Usmar, D. Rickwood, and M. T. Wilson, eds.) pp. 1-16. IRL Press, Oxford.
Srere, P. A. (1969). Citrate synthase. Methods Enzymol. 13, 3-11.
Trouet, A. (1974). Isolation of modified liver lysosomes. Methods Enzymol. 31, 323-329.
Trounce, I. A., Kim, Y. L., Jun, A. S., and Wallace, D. C. (1996). Assessment of mitochondrial oxidative phosphorylation in patient muscle biopsies, lymphoblasts, and transmitochondrial cell lines. Methods Enzymol. 264, 484-509.
Wharton, D. C., and Tzagoloff, A. (1967). Cytochrome oxidase from beef heart mitochondria. Methods Enzymol. 10, 245-250.
Whittaker, V. P. (1993). Thirty years of synaptosome research. J. Neurocytol. 22, 735-742.
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