[1] Overview—Preparation and properties of mitochondria from different sources

[1 ]

3

M I T O C H O N D R I A L P R E P A R A T I O N S A N D PROPERTIES

[1] O v e r v i e w - - P r e p a r a t i o n and Properties Mitochondria from Different Sources

By

JAN NEDERGAARD

and

BARBARA

of

CANNON

The majority of current research on mitochondria has been performed on mitochondria obtained from rat liver, rat heart, or ox heart since these mitochondria can be obtained readily and in quantity. After the development of conventional techniques for mitochondrial isolation, preparations were made from different tissues, but few attracted permanent interest and, as mitochondriology evolved, the liver mitochondrion was adopted as the '~typical mitochondrion," a position it hardly deserves. The accrued knowledge can now be used for the further study of mitochondria from other sources with varied functions and for studies of mitochondria in different stages of differentiation and in different pathological conditions. It is our aim in this article to provide references to recent papers on each preparation, and this has necessitated some historical injustice. The reader is also referred to the FASEB Handbook of Cell Biology (1976) for a more detailed tabulation of all enzymes and their localizations. The literature survey for this article was completed January 1977. Preparation Techniques All preparation methods demonstrate major similarities, but unfortunately the details are often tissue specific or laboratory specific. Little systematic investigation has been performed to motivate many of the methods.

Physiological State of the Organism The physiological state of the experimental organism must be carefully reported. Numerous controversial results from different laboratories can surely be explained in the light of physiological data.

Isolation Medium Principal Osmotic Support 1. Nonionic, e.g., sucrose, mannitol, or sorbitol or a mixture of these (mannitol has been claimed to adhere less to starch grains), about 250 mM for mammalian tissues. 2. Ionic, e.g., KCI (100-150 raM) for those tissues that assume a gelatinous consistency upon homogenization. There is, however, METHODS IN ENZYMOLOGY, VOL. LV

Copyright © 1979by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181955-8

4

ORGANELLES AND MEMBRANES

[1 ]

a risk that the high ionic strength may cause loss of certain peripheral proteins, e.g., cytochrome c, creatine kinase. 3. Ionic. Potassium aspartate (160 mM) has been successfully employed to obtain an isolation medium of more physiological nature. 1 Possible Additions

1. EDTA, or preferably EGTA (1 mM), in order to chelate Ca z+ ions, which can function as uncouplers and are also cofactors for certain phospholipases. 2. Bovine serum albumin (BSA) (0.1-1%), in order to bind free fatty acids, acyl-CoA esters, lysophospholipids, or other detergents. 3. A buffer (Tris, TES) 5-20 mM. 4. Dithiothreitol, cysteine, mercaptoethanol, or other sulfhydryl compounds to protect certain enzymes (0.1-0.5 mM). 5. Anticoagulants such as heparin or polyethylene sulfonate (PES) in order to prevent assumption of gelatinous consistency and agglutination in nonionic media (for muscle and brain). These components may be present in initial studies until a lack of effect is evident. Homogenization

Isolation of mitochondria necessitates the initial destruction of intercellular connections, cell walls, and plasma membranes by a means that as far as possible prevents damage to the intracellular organelles. If there is a great diversity of cells in a particular organ, mitochondrial isolation by homogenization will yield a heterogeneous preparation, the properties of which will represent the mean of the components. The selection of a destruction technique should be dictated by the type of cells and intercellular connections and is either mechanical or enzymatic. For soft tissues homogenization of a tissue mince using a Dounce hand homogenizer or a power-driven Potter-Elvehjem glass-Teflon homogenizer is often suitable. Grinding of the tissue with glass beads, a tissue or cell press, or a tissue blender (Polytron, UltraTurrax) may be employed in more obstinate cases. The ratio of tissue to isolation medium for homogenization is usually between 1 : 5 and 1 : 10 (w/v). Enzymatic destruction of cell walls and membranes by proteases is also common and occasionally inevitable, but a selective gradation of the enzyme action can be difficult to achieve. Temperatures close to 0 ° are used to minimize phos1E. N. Slack and E. Bursell, Biochim. Biophys. Acta 449, 491 (1976).

[1]

MITOCHONDRIAL PREPARATIONS AND PROPERTIES

5

pholipase and protease damage in mechanical procedures and also in some enzymatic methods. All subsequent operations are performed at 0o_4°"

Filtration Filtration of the homogenate through silk cloth (gauze, cheesecloth, Miracloth) removes unbroken tissue fragments, nerves, and connective tissue.

Differential Centrifugation If homogenization has been performed by enzymatic means or if the tissue has a high fat content, the homogenate is first centrifuged at high speed in order to rapidly separate the mitochondria from the noxious medium. The supernatant and possible fat cake are removed by rapidly inverting the tube, and the tube walls are wiped clean with a paper tissue. The pellet is resuspended in a minimal volume of medium, transferred to a clean tube, and diluted to the original volume, and the procedure described below is then carried out, commencing with low-speed centrifugation. Low-speed centrifugation is carried out to remove unbroken cells, cellular debris, and nuclei. Since mitochondria in the lower part of the centrifuge tube will also sediment in the pellet, a marginal increase in the yield can be obtained (after decanting the supernatant containing the major part of the mitochondria) by resuspending the pellet and recentrifuging at low speed. High-speed centrifugation of the combined supernatants is performed to sediment the mitochondria. However, lighter contaminants from the lower part of the tube are also sedimented and can be removed by resuspension of the pellet and recentrifugation. Because the sedimentation coefficients for mitochondria from the same tissue in different animals and especially for mitochondria from different tissues vary greatly, z the actual values, particularly of the highspeed centrifugation, must be determined for each new preparation. This can be done by measuring the distribution of cytochrome oxidase activity between pellet and supernatant in a series of centrifugations with increasing speed (5000-20,000 g) and determining at which speed no increased activity is found in the pellet. 2 To use values obtained from other tissues can lead to low yields or high contamination. Most laboratories use approximately 1000 g for 10 min for low-speed centrifugation and 10,000 g E. Slinde, E. Morild, and T. Flatmark, Anal. Biochem. 66, 151 (1975).

6

ORGANELLES AND MEMBRANES

[1]

for 10 min for high-speed centrifugation, and in this article this treatment is referred to as "routine centrifugations." The exact conditions may be obtained from the references. In many tissues a light-colored layer of fluffy material may be seen lying above the darker brown mitochondrial pellet after centrifugation. This should be carefully sloughed off by gentle shaking with a few drops of medium and removed, since it contains denatured proteins. In some tissues, two distinct mitochondrial fractions can be isolated. In all cases, the heavier one contains the metabolically most intact mitochondria. The lighter fraction is presumed to contain mitochondria that have been slightly damaged during preparation. Gradient Centrifugations

The pellet obtained as above should be considered " c r u d e , " and a purification based not only on size (S value) but also on density (p value) may be necessary. If desired, this should not be performed on a sucrose gradient since, in order to obtain higher densities, such high concentrations are required that the mitochondria may be damaged by the osmotic conditions, and the increase in the viscosity of the medium necessitates long centrifugation times. Ficoll (Pharmacia, Uppsala, Sweden) is a nonionic sucrose polymer that allows high densities to be obtained with small changes in osmolarity and viscosity. If there is doubt as to the purity of the " c r u d e " mitochondrial pellet, it may be layered on top of a continuous Ficoll gradient (5-35% in 250 mM sucrose) and the mitochondrial band identified. From its location a discontinuous Ficoll gradient can then be constructed to permit isolation of a pure fraction.3,4 Storage

Finally, the mitochondria should be suspended in a minimal volume of isolation medium. When ionic isolation media are employed, a nonionic storage medium may nevertheless be preferred to prevent loss of peripheral proteins. Certain mitochondria, during preparation in nonionic media, lose matrix ions and collapse because of lack of osmotic support. This results in low oxidation rates with NAD-linked substrates. For mitochondria in these situations, the final wash and suspension should be made in a medium of penetrant ions to facilitate regaining of the in situ volume. Addition of a phospholipase inhibitor (e.g., Nupercaine) has been successful in prolonging the metabolically active life of isolated D. W. McKeeland L. Jarett, J. Cell Biol. 44, 417 (1970). 4 L. Jarett, this series, Vol. 31 [4].

[1 ]

MITOCHONDRIAL PREPARATIONS AND PROPERTIES

7

mitochondria.5 Long-term storage of mitochondria in liquid nitrogen may occasionally be achieved. 6 Some General Mitochondrial Properties Mitochondria are adapted in different tissues for the function of the particular organ. Thus, the enzyme pattern and properties of mitochondria isolated from different sources will be expected to reflect the in vivo function of the organ. Nonetheless, certain general criteria should be satisfied in order to confirm the integrity of a mitochondrial preparation. Mitochondria are involved primarily in oxidative catabolism. Consequently, the majority of reactions can be followed by measuring oxygen consumption and complementing this with other analyses. Mitochondrial preparations are thus most often described and characterized by data obtained from oxygen electrode studies of respiration. Incubation Medium. For these studies the incubation medium may be nonionic or ionic but, in cases in which osmotic support is lost during preparation, ionic media should be chosen (e.g., 100 mM KCI). Other Additions 1. MgC12 (1-2 mM) for formation of ATP-Mg and ADP-Mg complexes and as enzyme cofactor. 2. Phosphate (5 mM), as counterion for malate uptake, and for oxidative phosphorylation. 3. Occasionally EDTA (1 mM). Some preparations contain an oligomycin-insensitive, Mg2+-dependent ATPase, which can be inhibited by chelating Mg z+. Respiratory control is then demonstrated by addition of an uncoupler. 4. Occasionally EGTA (1 mM) to chelate Ca z+, which may uncouple the mitochondria, but the resulting lack of Ca 2+ may be inhibitory to certain enzymes systems, r 5. Occasionally BSA (0.1-1%) to remove fatty acids and their esters, which inhibit adenine nucleotide translocases, tricarboxylate carriers, and other enzymes and can also function as uncouplers. Substrates 1. Citric acid cycle intermediates are oxidized by most mitochondria, but a counterion for the permeases, most often 5 mM A. Scarpa and J. G. Lindsay, Eur. J. Biochern. 27, 401 (1972). 6 S. Fleischer and M. Kervina, this series, Vol. 31 [2]. r K. Malmstrrm and E. Carafoli, Biochem. Biophys. Res. Cornmun. 69, 658 (1976).

8

ORGANELLES AND MEMBRANES

[1]

malate, must be present. Malate will then also function as a condensing partner when necessary (for pyruvate and fatty acid respiration). 2. Fatty acids should be added as the carnitine ester or in the presence of ATP, CoASH, and carnitine. Nagarse-treated mitochondria lose long-chain acyl-CoA synthetase activity, and fatty acids cannot therefore be used as substrates. 3. For mammalian mitochondria in which no external NADH dehydrogenase is found, an indication of membrane integrity is often taken as a very low rate of respiration with added NADH as substrate. 4. Other possible substrates include 3-hydroxybutyrate, glutamate, pyruvate, proline, and glycerol 3-phosphate.

Criteria o f Integrity The respiratory control ratio (RCR) is defined as follows: 5 [ respiration rate in the presence of added substrate and phosphate acceptor(ADP)]/ (rate after consumption of ADP), i.e., (State 3/State 4). High values indicate that the mitochondria have a good energy-conserving capacity. Low values, however, should be viewed with caution. They may indeed indicate that a large energy leakage is occurring in State 4. Alternatively, they may demonstrate the use of an unsuitable substrate (e.g., no substrate permease), that the substrate is being used under incorrect conditions (e.g., no counterion), or that ADP translocation is rate limiting. In all cases there is little energy leakage but there is a severe rate limitation in State 3. An absolute value (nmoles oxygen per minute and milligram protein) of State 4 can in certain systems be more informative than RCR, although some stimulation with ADP or an uncoupler must also be demonstrated. The ADP/O ratio (P/O ratio) is defined as follows: (total amount of ADP added)/(amount of oxygen consumed from the addition of ADP until the respiration returns to a low rate). State 4 respiration should not be subtracted. The ATP/ADP ratio varies during the cycle from very low to infinitely high. The P/O ratio can also be measured using glucose plus hexokinase to regenerate ADP, by this means maintaining a very low ATP/ADP ratio, which results in higher P/O ratios than in the first case. Respiration-linked Ca 2+ accumulation and high-affinity Ca 2+ binding have been demonstrated in all vertebrate tissues so far studied.S'9 They have also been shown in cicada but not in blowfly, where only a slow, nonstoichiometric Ca 2+ accumulation is found. This is also the case in s A. L. L e h n i n g e r , Biochem. J. 119, 129 (1970). 9 E. Carafoli and A. L. Lehninger, Biochem. J. 122, 681 (1971),

[1]

MITOCHONDRIAL PREPARATIONS AND PROPERTIES

9

Neurospora crassa. Saccharomyces cerevisiae and Candida utilis have no accumulation and no high-affinity sites. Maize seedlings, white potato, and sweet potato show respiration-linked accumulation, but the presence of high-affinity sites has not been investigated. Five different ATP-dependent acyl-CoA synthetases have been reported. The chain length optima for these are C2, C4, C7, C,2, and C,6. The first three are localized in the mitochondrial matrix, whereas the two long-chain synthetases are found on the outer membrane (and on microsomes). Liver, kidney, and heart mitochondria possess all five enzymes; brain and testis lack C2; adrenal cortex lacks C2 and C4; and striated muscle and epidydimal fat lack C~, C4, and C7. Long-chain activation is most active, except in kidney, where propionate is activated at the highest rate. In relation to the protein content of the tissue, the highest specific activity of the long-chain synthetase is found in epidydimal fat.'° Substrate Permeases (see, e.g., Meijer and van DamH). Liver mitochondria are known to possess at least the following permeases: (1) hydroxyl/ phosphate, (2) pho sphate/malate/succinate/oxaloacetate, (3)malate/ 2-oxoglutarate, (4) malate/citrate/isocitrate/phosphoenolpyruvate, (5) hydroxyl/glutamate, (6) glutamate/aspartate, (7) hydroxyl?/pyruvate. Most mitochondria possess most of these, but the permeases are seldom studied as such. However, it should be noted that (5) is exclusive for liver; heart muscle lacks (4) and has (6); brain has (7); yeast has (7), but this can also transport lactate; blowfly lacks (2), (3), and (4), i.e., all citric acid cycle intermediate permeases; and kidney has (6) and also a glutamate/glutamine permease. Mitochondrial DNA is generally circular. In animals it is approximately 5 /~m long; in plants, 35 /zm; in Saccharomyces, 25 /zm; and in Neurospora, 20/zm.'2 Mitochondrial Preparations MAMMALIAN TISSUES Adrenal Cortex The mitochondrial suspension is prepared from bovine adrenal cortex or from rat adrenal glands in buffered sucrose-EDTAlZ-16; BSA is also 1o M. Aas, Biochim. Biophys. Acta 231, 32 (1971). 11 A. J. Meijer and K. v a n Dam, Biochim. Biophys. Acta 346, 213 (1974). 12 p. Borst and R. A. Flavell, Handb. Biochem. Mol. Biol., 3rd Ed. 2, 363 (1976). 13 D. R. Pfeiffer and T. T. T. Chen, Biochem. Biophys. Res. Cornmun. 50, 807 (1973). 14 E. R. Smith and D. L. Williams-Smith, Biochirn. Biophys. Acta 404, 309 (1975). 15 F. G. Peron, C. P. W. Tsang, and A. Haksar, Biochim. Biophys. Acta 270, 266 (1972). 16 L. A. Sauer and P. J. Mulrow, Arch. Biochem. Biophys. 134, 486 (1969).

10

ORGANELLES AND MEMBRANES

[1]

recommended. 13,14,16 Dithiothreitol can be used to preserve cytochrome P-450 and NADPH-adrenodoxin reductase.17 The mitochondria are released using a glass-Teflon homogenizer and collected by routine centrifugations. 14,15 Yield: bovine, 10 mg protein per gram wet weight; 0.5 mg protein per rat.

Properties. The mitochondria possess a normal respiratory chain 13 and oxidize citric acid cycle intermediates with fair respiratory control and expected P/O values.IS,16 Energy-dependent Ca 2+ accumulation can occur. 14,19The ultrastructure of the mitochondria is unusual; the matrix contains vesicular inner structures possibly separated from the inner membrane.2°,21 The mitochondria contain three monooxygenase systems involved in corticosteroidogenesis; lift-and 18-hydroxylation of deoxycorticosterone (DOC) to corticosterone and side chain cleavage of cholesterol to pregnenolone. ~2"28These involve a second electron-transport chain, in which the primary electron donor is NADPH.22,23 Electrons are then transferred to a flavoprotein, NADPH-adrenodoxin reductase, to adrenodoxin (an iron-sulfur compound similar to ferredoxin), and to cytochrome P-450, where hydroxylation of the steroid precursor occurs. 22,23 The two electron-transport chains are not independent, and steroid hydroxylation can be shown to inhibit ATP synthesis.18"2°_ Hydroxylation of DOC stimulates oxygen consumption and can give apparent transitions from State 4 to State 3.16"20_Depending on the substrate, NADPH is supplied either by action of the energy-linked transhydrogenase or from the " m a l i c " enzyme that converts malate to pyruvate, the latter being formed stoichiometrically with DOC hydroxylation. 13-16,19,22-24 Deoxycorticosterone must penetrate the inner membrane to the site of 1 lft-hydroxylation, and it is suggested that this site is possibly on the vesicular inner structures. 2° Two entirely independent forms of cytochrome P-450 occur, one involved in 11/3- and 18-hydroxylation and one in side chain cleavage.14,17,22,25Adrenocorticotropin, which is involved in growth and proliferation of the mitochondria, 26also increases association 17 I. Bj6rkhem and K-E. Karlmar, Eur. J. Biochem. 51, 145 (1975). 18 W. C a m m e r a n d R. W. Estabrook, Arch. Biochem. Biophys. 122, 721 (1967). ~9 D. R. Pfeiffer and T. T. T. Chen, Biochemistry 14, 98 (1975). 2o j. R y d s t r 6 m , J. •. G u s t a f s s o n , M. Sundberg-Ingelman, J. Montelius, and L. Ernster, Biochem. Biophys. Res. Commun. 73, 555 (1976). 21 j. D. Lever, J. Biophys. Biochem. Cytol. 2, 313 (1956). 22 E. R. S i m p s o n and G. S. Boyd, Eur. J. Biochem. 22, 489 (1971). 23 E. R. S i m p s o n and R. W. Estabrook, Arch. Biochem. Biophys. 129, 384 (1969). z4 S. B. O l d h a m , J. J. Bell, and B. W. Harding, Arch. Biochem. Biophys. 123, 496 (1968). ~5 C. R. Jefcoate, W. H. O r m e - J o h n s o n , and H. Beinert, J. Biol. Chem. 251, 3707 (1976). 28 G. G. N u s s d o r f e r , G. Muzzacchi, P. Rebuffat, A. S. Belloni, G. Gottardo, and A. M. G a m b i n o , Cell Tissue Res. 163, 273 (1975).

[1]

MITOCHONDRIAL PREPARATIONS AND PROPERTIES

11

of cholesterol with c y t o c h r o m e P-450 and thus stimulates side chain cleavage. 14 High Ca z+ concentrations mimic to some extent this effect. 14 Calcium ion is also required for action of the " m a l i c " enzyme. 13,19 Cyt o c h r o m e P-450 occurs in up to 10-fold excess over c y t o c h r o m e oxidase.~S'27'2SAdrenodoxin has not been found in crude adrenal mitochondrial preparations from the tiger shark and green sea turtle, although c y t o c h r o m e P-450 occurs there.27 Testis The mitochondria are prepared from male rats by isolation in sucrose, homogenization (Teflon pestle), and routine centrifugations. The final suspension is in KCI. P r o p e r t i e s . The inner membrane has a 3fl-hydroxysteroid dehydrogenase, which takes part in the synthesis of-steroid hormones (testosterone). 29 The mitochondria are claimed to possess an adenylate cyclase, which is stimulated by human chorionic gonadotropin.30

K i d n e y Cortex The mitochondrial suspension is prepared from chicken, pig, or rat in buffered mannitol or sucrose with E D T A by routine procedures. 31,32Both inner and outer medulla are discarded. Dithiothreitol may also be included. 32 P r o p e r t i e s . The mitochondria oxidize citrate, succinate, 2-0xoglutarate, malate, 3-hydroxybutyrate, pyruvate, 31 and glutamine and glutamate 31"33 but can show poor respiratory control due to an oligomycininsensitive Mg2÷-dependent ATPase in the outer membrane. 34 They form oxaloacetate from CO2 and pyruvate.35 Kidney mitochondria convert 25OH-cholecalciferol (25-OH-vitamin D3) to 1,25-(OH)2-cholecalciferol, the active vitamin D hormone, by a m o n o o x y g e n a s e system through an

27T. Kimura, H. W. Ping, L. R. de Alvare, K. V. Honn, and W. Chavin, Comp. Biochem. Physiol. 53B, 273 (1976). 2s H. W. Ping and T. Kimura, Biochim. Biophys. Acta 423, 374 (1976). 29S. Solimovici, B. Bartoov, and B. Lunenfeld, Biochim. Biophys. Acta 321, 27 (1973). 30S. Solimovici, B. Bartoov, and B. Lunenfeld, Biochim. Biophys. Acta 377, 454 (1975). 31M. W. Weiner and H. A. Lardy, J. Biol. Chem. 248, 7682 (1973). 32j. G. Ghazarian, C. R. Jefcoate, J. C. Knutson, W. H. Orme-Johnson, and H. F. DeLuca, J. Biol. Chem. 249, 3026 (1974). 33j. C. Knutson and H. F. Deluca, Biochemistry 13, 1543 (1974). 34p. Gmay, C. Nowicka, and S. Angielsky, FEBS Lett. 47, 76 (1974). 35M. A. Mehlman, J. Biol. Chem. 243, 3289 (1968).

12

ORGANELLES AND MEMBRANES

[1]

electron-transport chain: NADPH ~ renal ferredoxin reductase ~ renal f e r r e d o x i n ---, c y t o c h r o m e P-450. 36 C y t o c h r o m e P - 4 5 0 h a s as y e t b e e n c l e a r l y d e m o n s t r a t e d o n l y in c h i c k k i d n e y m i t o c h o n d r i a . 32 T h e o x y g e n a s e s y s t e m c a n b e i n d u c e d b y l o w a m o u n t s o f C a 2+ in t h e diet. 32 I n t r a c e l l u l a r C a 2+ o r p h o s p h a t e m a y r e g u l a t e the 1 - h y d r o x y l a s e a c t i v i t y . 37 A 24-hydroxylation has also been demonstrated, but the function of the product is u n k n o w n . 33 T h e s e s t u d i e s h a v e h a d to b e c a r r i e d o u t o n c h i c k e n k i d n e y , s i n c e in v i t r o s t u d i e s in r a t a r e p r e v e n t e d b y t h e p r e s e n c e o f an i n h i b i t o r y f a c t o r in t h e h o m o g e n a t e , as T h e m i t o c h o n d r i a a l s o p r o d u c e a m m o n i a a n d g l u t a m a t e f r o m g l u t a m i n e b y t h e p h o s p h a t e - d e p e n d e n t glut a m i n a s e . T h i s a m m o n i a is i n v o l v e d in p r o t o n e x c r e t i o n in t h e u r i n e . 39 A g l u t a m i n e - g l u t a m a t e a n t i p o r t e r h a s b e e n s u g g e s t e d , 4° b u t p h o s p h a t e t r a n s p o r t is a l s o i n v o l v e d in g l u t a m i n e u p t a k e . 41 G l u t a m i n e t r a n s p o r t is i n h i b i t e d b y 2 - 0 x o g l u t a r a t e . 42 C h i c k e n s , w h i c h e x c r e t e uric a c i d a n d n o t u r e a , h a v e n o a r g i n a s e in t h e l i v e r b u t o n l y in t h e k i d n e y m i t o c h o n d r i a l m a t r i x . 43 H i g h b i c a r b o n a t e c o n c e n t r a t i o n s i n f l u e n c e c i t r a t e t r a n s p o r t o v e r t h e m i t o c h o n d r i a l m e m b r a n e , a n d this h a s b e e n r e l a t e d to t h e i n c r e a s e d c i t r a t e c l e a r a n c e in m e t a b o l i c a l k a l o s i s . 44 T h e m i t o c h o n d r i a c o n t a i n an L2 - h y d r o x y a c i d o x i d a s e o f u n k n o w n p h y s i o l o g i c a l function.45

Striated Muscle T h e m i t o c h o n d r i a l s u s p e n s i o n is p r e p a r e d f r o m r a t ( m u s c u l i g a s t r o c n e m i u s , m a s s e t e r , v a s t u s ) , d o g h i n d leg, o x n e c k , h u m a n b i o p s y s a m p l e , o r p i g e o n b r e a s t 46-5° in b u f f e r e d K C 1 - E D T A c o n t a i n i n g A T P a n d 3e j. I. Pedersen, J. G. Ghazarian, N. R. Orme-Johnson, and H. F. DeLuca, J. Biol. Chem. 251, 3933 (1976). 37 H. L. Henry and A. W. Norman, Arch. Biochem. Biophys. 172, 582 (1976). as K. M. Botham, J. G. Ghazarian, B. E. Kream, and H. F. DeLuca, Biochemistry 15, 2130 (1976). 30 N. P. Curthoys, T. Kuelenschmidt, and S. S. Godfrey, Arch. Biochem. Biophys. 174, 82 (1976). 40 M. Crompton and J. B. Chappell, Biochem. J. 132, 35 (1973). 41 Z. Kovarevir, Biochim. Biophys. Acta 430, 399 (1976). 42 L. Goldstein, Biochem. Biophys. Res. Commun. 70, 1136 (1976). 43 H. Kodawaki, H. W. Israel, and M. C. Nesheim, Biochim. Biophys. Acta 437, 158 (1976). 44 D. P. Simpson and S. Angielsky, Biochim. Biophys. Acta 298, 115 (1973). 45 C. E. Domenech, E. E. Machado de Domenech, and A. Blanco, Biochim. Biophys. Acta 321, 54 (1973). 46 S. R. Max, J. Garbus, and H. J. Wehman, Anal. Biochem. 46, 576 (1972). 47 G. F. Azzone and E. Carafoli, Exp. Cell Res. 21,447 (1960). 4s M. W. Makinen and C.-P. Lee, Arch. Biochem. Biophys. 126, 75 (1968). 49 K. S. Cheah, Biochim. Biophys. Acta 275, 1 (1972). 50 p. H. E. Groot and W. C. Hfilsmann, Biochim. Biophys. Acta 316, 124 (1973).

[1 ]

MITOCHONDRIAL PREPARATIONS AND PROPERTIES

13

MgCI2, 4s'5°-53 or in buffered m a n n i t o l - s u c r o s e - E D T A 54"55 or in sucrose with heparin. 46"s6 An ionic m e d i u m or heparin prevents the assumption of a gelatinous consistency and mitochondrial agglutination during homogenization.46"48"50-53'56 Heparin also p r e v e n t s the action of a deleterious lipoprotein lipase. 46 Albumin is beneficial during isolation in the absence of heparin. 4s.5~ The mitochondria are isolated by mechanical cell disruption (glass-Teflon homogenizer, UltraTurrax, glass beads, etc. 46,48,50,52,57), or by e n z y m a t i c digestion [trypsin, B. subtilis proteinase (Nagarse) 4s's°'s4"ss'sr'ss] and subsequent centrifugations. There is often lysosomal e n z y m e contamination. 46 Further purification can be p e r f o r m e d on a sucrose density gradient, ss Yield: 2-4 mg protein per gram wet weight. Properties. The mitochondria oxidize glutamate, 2-0xoglutarate, succinate, p y r u v a t e plus malate, and medium- and long-chain fatty acids (plus malate, ATP, C o A S H , and carnitine). 46"5° Citrate p e r m e a s e m a y be lacking. 46 O x a l o a c e t a t e d e c a r b o x y l a s e is absent. ~9 N o oxidation is found with fumarate or 3-hydroxybutyrate.46 Glycerol 3-phosphate oxidation is induced disproportionately in hyperthyroidism. 6° Activation of long- and medium-chain fatty acids is on the same m e m b r a n e - b o u n d e n z y m e , in contrast to heart, k i d n e y , and liver, where medium- and short-chainactivating e n z y m e s are in the matrix. S° The c y t o c h r o m e content is norreal. 48 Oxidation rates up to 300 nmol O per minute and milligram protein are found with palmitate as substrate. ~° G o o d RCR and e x p e c t e d P/O ratios are observed. 46"49,54"5s A very minor fraction of creatine kinase, although with significant activity, resides in the mitochondria.61

H e a r t M u s c l e (see also, this v o l u m e , Articles [4] a n d [15]) The mitochondrial suspension is p r e p a r e d from ox, rabbit, rat, or guinea pig in buffered s u c r o s e - E D T A or m a n n i t o l - s u c r o s e - E D T A using 51 j. B. Chappell and S. V. Perry, Nature (London) 173, 1094 (1954). 52 j. B. Peter and L. D. Lee, Biochem. Biophys. Res. Commun. 29, 430 (1967). 53 L. E r n s t e r and K. N o r d e n b r a n d , this series, Vol. 10 [14]. 54 G. Bullock, E. E. Carter, and A. M. White, FEBS Lett. 8, 109 (1970). 55 j. H. Thakar, K. W r o g e m a n n , and M. C. Blanchaer, Biochim. Biophys. Acta 314, 8

(1973). 56D. S. Dow, Biochemistry 6, 2915 (1967). 5r j. j. Mockel and D. S. Beattie, Arch. Biochem. Biophys. 167, 301 (1975). .~sG. R. Bullock, E. E. Carter, and A. M. White, Biochim. Biophys. Acta 292, 350 (1973). 59A. B. Wojtczak and E. Wa~ajtys, Biochirn. Biophys. Acta 347, 168 (1974). 6oj. R. Tata, L. Ernster, O. Lindberg, E. Arrhenius, S. Pedersen, and R. Hedman, Biochem. J. 86, 408 (1963). 61H. R. Scholte, Biochim. Biophys. Acta 305, 413 (1973).

14

ORGANELLES AND MEMBRANES

[1]

mechanical homogenization (UltraTurrax, Polytron PT10S2.e3)orNagarse digestion,~4-6efollowed by routine centrifugations with a considerable number of washes to remove the lighted layer.

Properties. The mitochondria are primarily catabolic and have densely packed cristae and a high cytochrome content. The major substrate for mammalian heart in vivo is fatty acids, 88and the mitochondria contain all five activating enzymes. 10 Palmitoyl carnitine plus malate and pyruvate plus malate are oxidized rapidly with high RCR and expected P/O ratios. In contrast to liver, in rat heart/3-0xidation limits the rate of palmitate oxidation. 68In the resting state the citric acid cycle is controlled at citrate synthase and at 2-0xoglutarate dehydrogenase. 67,68Exogenous citrate and isocitrate are not oxidized since the tricarboxylate carrier has low activity. 69 No glutamate-hydroxyl exchange exists; there is only a glutamateaspartate translocator, and glutamate dehydrogenase activity is very low. 70,71There is rapid Ca 2+ uptake and high-affinity binding. 63,72The role of mitochondrial Ca 2+ uptake in the regulation of the beat-to-beat Ca 2+ cycle is uncertain, ~ although certain kinetic data are compatible with its involvement.72 A Na+-Ca 2+ exchange has also been reported.73 Twenty percent of the tissue creatine kinase activity is mitochondrial and found on the outer surface of the inner membrane.61 Association of the inner and outer membranes has been suggested to involve peptide bonding. TM An outer membrane oligomycin-insensitive Mg2+-stimulated ATPase has been reported. 75 Smooth Muscle (see also, this volume, Article [7]) The mitochondrial suspension is prepared from human myometrium, bovine main pulmonary artery, or mesenteric vein in buffered sucrose TM e2 I. Vallin, Biochim. Biophys. Acta 162, 477 (1968). A. Scarpa and P. Graziotti, J. Gen Physiol. 62, 756 (1973). e4 B. Chance and B. Hagihara, Prec. Int. Congr. Biochem., 5th, 1961, Vol. 5, p. 3 (1963). es S. V. Pande and M. C. Blanchaer, J. Biol. Chem. 246, 402 (1971). e~ R. G. Hansford and R. N. Johnson, J. Biol. Chem. 250, 8361 (1975). 6r K. F. LaNoue, J. Bryla, and J. R. Williamson, J. Biol. Chem. 247, 667 (1972). 68 K. F. LaNoue, E. I. Walajtys, and J. R. Williamson, J. Biol. Chem. 248, 7171 (1973). e9 S. Cheema-Dhadli, B. H. Robinson and M. L. Halperin, Can. J. Biochem. 54, 561 (1976). Toj. B. Chappell, Br. Med. Bull. 24, 150 (1968). 71 E. J. Davis, Biochim. Biophys. Acta 162, 1 (1968). r2 M. Crompton, E. Sigel, M. Salzmann, and E. Carafoli, Eur. J. Biochem. 69, 429 (1976). r3 M. Crompton, M. Capano, and E. Carafoli, Eur. J. Biochem. 69, 453 (1976). ~4 H. R. Scholte, Biochim. Biophys. Acta 330, 283 (1973). r~ E. K. Vijayakumar and M. J. Weidemann, Biochem. J. 160, 383 (1976). 7~ S. Batra, Biochim. Biophys. Acta 305, 428 (1973).

[1]

MITOCHONDRIAL PREPARATIONS AND PROPERTIES

15

or buffered KCI-EDTA containing ATP, MgSO4, and BSA r7 by homogenization (glass-Teflon TM or Polytron PTI0 rT) or by enzymatic digestion with Nagarse r7 and subsequent routine centrifugations. Nagarse treatment yields less well coupled mitochondria. In all cases, BSA greatly improves respiratory control. Further purification can be performed on a sucrose density gradient. 77

Properties. The mitochondria show good RCR and expected P/O ratios with glutamate plus malate, pyruvate plus malate, succinate, and glycerol 3-phosphate. 77 There is a high Ca 2÷ content and a rapid respiration-supported uptake (12 nmol mg -1 sec-1).76"77 The mitochondria can accumulate up to 210 nmol Ca z+ per milligram protein without uncoupling. In contrast to striated muscle, smooth muscle contains little sarcoplasmic reticulum, and it is therefore feasible that the mitochondria act as regulators of cytoplasmic C a 2 + . 77 White Adipose Tissue The mitochondrial suspension is prepared from rat epidydimal fat pads by homogenization (Polytron PT20) 7s or from fat cells (obtained by collagenase treatment of the fat pads) by vortexing for 1 min in sucroseBSA-glutathione-EGTA. TM Isolated by rapid centrifugations (20,000 g, 1 min) s° or by routine procedures sl or from an adipocyte fractionation procedure using discontinuous Ficoll gradients. 4

Properties. The mitochondria oxidize palmitoyl carnitine,79 succinate, pyruvate, 2-0xoglutarate (all plus malate) with acceptable RCR and P/O ratios. The rate of glycerol 3-phosphate oxidation is low, especially when compared with brown adipose tissue mitochondria. The white adipose tissue mitochondria do not oxidize acetate and oxidize acetyl carnitine only slowly, s° No NADH oxidation is found, s2 The tri- and dicarboxylic acid permeases are similar to the ones found in rat liver. 8° The mitochondria play an important role in lipogenesis. Pyruvate gives rise to citrate and malate in the presence of carbonate, 79 but pyruvate plus carbonate does not stimulate palmitoyl carnitine oxidation as much as added malate.S2 Carnitine does not increase palmitoyl carnitine oxidation in the absence of malate, which implies that carnitine cannot act as an 7T j. Valli6res, A. Scarpa, and A. P. Somlyo, Arch. Biochem. Biophys. 170, 659 (1975). 7s D. L. Severson, R. M. Denton, B. J. Bridges, and P. J. Randle, Biochem. J. 154, 209 (1976). 79 B. R. Martin and R. M. Denton, Biochem. J. 125, 105 (197J). so B. R. Martin and R. M. Denton, Biochem. J. 117, 861 (1970). sl M. S. Patel and R. W. H a n s o n , J. Biol. Chem. 245, 1302 (1970). s2 R. D. H a r p e r and E. D. Saggersson, Biochem. J. 152, 485 (1975).

16

ORGANELLES AND MEMBRANES

[1]

" a c e t a t e sink" forming acetyl carnitine, s2 This is in contrast to brown adipose tissue mitochondria. Acetyl carnitine can, however, be formed from pyruvate and carnitine. 79 The rate of oxidation o f palmitoyl-CoA plus carnitine is much slower than that of palmitoyl carnitine, and the transferase may be rate limiting and direct the acyl groups toward esterification. 82 In lipogenesis, citrate leaves the mitochondria, donates acetylC o A to the cytoplasm, and returns as malate or pyruvate. When necessary, N A D P H can also be formed in the cytoplasm from citrate or through the m a l a t e - p y r u v a t e shuttle, s° Pyruvate dehydrogenase activity is increased by insulin.S3 The regulation of pyruvate dehydrogenase phosphatase and kinase is currently being studied. TM Calcium ion is rapidly accumulated in the mitochondria, but changes in Ca 2+ concentration do not seem to be the physiological regulator of pyruvate dehydrogenase.78 B r o w n A d i p o s e T i s s u e (see also, this v o l u m e , Article [8]) The mitochondrial suspension is prepared from n e w b o r n or coldadapted rat, rabbit, hamster, or guinea pig in buffered sucrose by homogenization (glass-Teflon) followed by a high-speed centrifugation, after which the fat cake is removed. Then routine centrifugations are performed. 84--86The final suspension is in KCI. 87 P r o p e r t i e s . A purine nucleotide (often GDP) and BSA must generally be present in the incubation medium in order to demonstrate energy conservation, s6"ss's9 The mitochondria oxidize palmitoyl carnitine plus malate very rapidly and palmitate (plus malate, ATP, carnitine, and CoA) with the same speed, in contrast to white adipose tissue mitochondria. Carnitine can function as an acetyl sink, and hamster and lamb mitochondria have an a c e t y l - C o A hydrolase, permitting fatty acid oxidation at high rates in the absence of malate.9°'91 Glycerol 3-phosphate oxidation rates are also very high, but citric acid cycle intermediates often

83D. L. Severson, R. M. Denton, H. T. Pask, and P. J. Randle, Biochem. J. 140, 225 (1974). s4 K. J. Hittelman, O. Lindberg, and B. Cannon, Eur. J. Biochem. 11, 183 (1969). s5 T. Flatmark and J. Pedersen, Biochim. Biophys. Acta 416, 53 (1975). sn j. Rafael, D. Klaas, and H.-J. Hohorst, Hoppe-Seyler's Z. Physiol. Chem. 349, 1711 (1968). sr D. G. Nichoils, H. J. Gray, and O. Lindberg, Eur. J. Biochem. 31, 526 (1972). as E. N. Christiansen, J. I. Pedersen, and H. J. Grav, Nature (London) 222, 857 (1969). s~ B. Cannon, D. G. Nicholls, and O. Lindberg, in "Mechanisms in Bioenergetics" (G. F. Azzone et al., eds.), p. 357. Academic Press, New York, 1973. 90B. Cannon, L. Romert, U. Sundin, and T. Barnard, Comp. Biochem. Physiol. 56B, 87 (1977). 91V. S. M. Bernson and D. G. Nicholls, Eur. J. Biochem. 47, 517 (1974).

[1]

M I T O C H O N D R I A L P R E P A R A T I O N S A N D PROPERTIES

17

s h o w low oxidation rates.9°'92 M i t o c h o n d r i a l A T P s y n t h e t a s e is v e r y low w h e n c o m p a r e d to r e s p i r a t o r y chain c a p a c i t y ( e x c e p t in lamb). 9°'93 The m i t o c h o n d r i a have unusually high anion permeabilities, w h i c h can be diminished b y purine nucleotides. 94 T h e m i t o c h o n d r i a are the site o f heat p r o d u c t i o n in c a t e c h o l a m i n e - i n d u c e d thermogenesis.95,96 B r a i n (see also, this v o l u m e , Article [6]) Nonsynaptic

Origin

T h e m i t o c h o n d r i a l s u s p e n s i o n is c o m m o n l y p r e p a r e d f r o m rat cerebral h e m i s p h e r e s 97-1°z in a buffered s u c r o s e - E D T A m e d i u m using a D o u n c e h o m o g e n i z e r 9s or, alternatively, by proteinase digestion and h o m o g e n i zation. 9r W h e n proteinase is used, B S A is required. 9r T h e p r o d u c t o f differential centrifugation also contains myelin, n e r v e endings, and synaptic vesicles. T h e m i t o c h o n d r i a are purified on a Ficoll gradient 98 and finally s u s p e n d e d in i s o o s m o t i c mannitol, s u c r o s e , or KCI. T h e yield is 3 - 4 mg protein per brain. P r o p e r t i e s . P o t a s s i u m chloride (20-30 mM) is essential for substrate oxidation for all substrates e x c e p t glycerol 3-phosphate. 9s This is p e r h a p s related to matrix expansion. Substrate t r a n s l o c a s e s are similar to those in liver, a l t h o u g h there is no g l u t a m a t e - h y d r o x y l e x c h a n g e , lO1,1o2,104Separate p y r u v a t e and 3 - h y d r o x y b u t y r a t e t r a n s l o c a s e s are present.l°° T h e r e are v e r y active acetyl- and a c y l - C o A h y d r o l a s e s , w h i c h are possibly mitochondrial, lO5 N o r m a l R C R and P/O values h a v e b e e n r e p o r t e d . In i m m a t u r e rat brain, r e s p i r a t o r y rates increase f r o m 1 to 3 w e e k s and then decline again f r o m 3 to 5 weeks.'°3 Creatine kinase is p r e s e n t m i t o c h o n drially.l°n H e x o k i n a s e is associated with the m i t o c h o n d r i a l m e m b r a n e , and glucose therefore acts as a p h o s p h a t e a c c e p t o r . 97 G l u c o s e 6-phos-

92j. Hou~t6k, B. Cannon, and O. Lindberg, Eur. J. Biochem. 54, 11 (1975). 93B. Cannon and G. Vogel, FEBS Lett. 76, 284 (1977). 94 D. G. NichoUs and O. Lindberg, Eur. J. Biochem. 37, 523 (1973). 95O. Lindberg, "Brown Adipose Tissue." Am. Elsevier, New York, 1970. 9e L. Jansk~, Biol. Rev. Cambridge Philos. Soc. 48, 85 (1973). 97 C. L. Moore and F. F. J6bsis, Arch. Biochem. Biophys. 138, 295 (1970). 98j. B. Clark and W. J. Nicklas, J. Biol. Chem. 245, 4721 (1970). 99j. M. Walsh and J. B. Clark, J. Neurochem. 26, 1307 (1976). lo0j. M. Land, J. Mowbray, and J. B. Clark, J. Neurochem. 26, 823 (1976). l°1S. C. Dennis, J. M. Land, and J. B. Clark, Biochem. J. 156, 323 (1976). 1°2M. D. Brand and J. B. Chappell, Biochem. J. 140, 205 (1974). '°aD. Holtzman and C. L. Moore, J. Neurochem. 24, 1011 (1975). ~°4A. Minn, J. Gayet, and P. Delorme, J. Neurochem. 24, 149 (1975). l°SA. H. Koeppen, E. J. Mitzen, and A. A. Ammoumi, Biochemistry 13, 3589 (1974). 1°6T. Sugano and O. Nagai, J. Biochem. (Tokyo) 70, 417 (1971).

18

OROANELLESAND UEMBaAr~ES

[1]

phate releases the enzyme, a7 A Crabtree effect can be demonstrated, although it is probably not physiological. 97 3,-Aminobutyric acid stimulates ATP synthesis with glutamate present. 107There is an active pyruvate carboxylase, x0s

Synaptosomal A crude mitochondrial fraction is isolated from a brain homogenate in 0.32 M sucrose and layered onto a Ficoll gradient. " F r e e " mitochondria form the pellet, and synaptosomes are collected at the interface. They are lysed osmotically in 6 mM Tris-C1, pH 8, for 60 min at 4 ° and centrifuged. The mitochondrial pellet is taken up in a 3% Ficoll medium and washed in a medium containing BSA. The resulting mitochondria are well coupled. Pyruvate plus malate gives good respiration, and pyruvate dehydrogenase is relatively high compared with " f r e e " mitochondria, whereas glutamate dehydrogenase is relatively low. 109 Protein synthesis has been reported in synaptosomal mitochondria.11°

Thyroid The mitochondrial suspension is prepared from sheep, rat, ox, or man in buffered mannitol-sucrose-EDTA, 111-11~occasionally containing ATP and BSA, 112,113either by Nagarse digestion and gentle homogenization re,n2 or mechanically in a glass-Teflon homogenizer 113and followed by routine centrifugations. Yield: 0.5-1.5 mg protein per gram wet weight.

Properties. The mitochondria oxidize citric acid cycle intermediates and pyruvatem-113; human mitochondria also oxidize 3-hydroxybutyrate and caproate. 11~ Fair RCR and expected P/O ratios have been obtained.Ill-113 Adenylate kinase activity is high, and both added AMP and ADP can therefore function as phosphate acceptors.111 The amount of pyridine nucleotides, especially NADP, is low in sheep compared with rat liver, although the ratio of NADP to NAD is as in heart, kidney, and brain.113 Thyroxine inhibits State 3 respiration. 113 l°7L.-W. Lee, C.-L. Liao, and F. M. Yatsu, J. Neurochem. 23, 721 (1974). 1°8M. S. Patel and S. M. Tilghman, Biochem. J. 132, 185 (1973). 109j. C. K. Lai and J. B. Clark, Biochem. J. 154, 423 (1976). H°H. B. Bosmann and B. A. Hemsworth, J. Biol. Chem. 245, 363 (1970). HaD. D. Tyler and J. Gonze, this series, Vol. 10 [17]. "2B. Inatsuki, M. Hiraga, and F. K. Anan, J. Biochem. (Tokyo) 74, 837 (1973). "aF. M. Lamy, F.oR. Rodesch, and J. E. Dumont, Exp. CellRes. 46, 518 (1967).

[1]

MITOCHONDRIAL PREPARATIONS AND PROPERTIES

19

Thymus T h e m i t o c h o n d r i a l s u s p e n s i o n is p r e p a r e d f r o m c a l f o r r a t in m a n n i t o l sucrose-EDTA by glass-Teflon homogenization and routine centrifugat i o n s . 114-116

P r o p e r t i e s . T h e m i t o c h o n d r i a h a v e b e e n s t u d i e d p r i n c i p a l l y in c o n n e c t i o n w i t h t h y m i c n u c l e a r m e m b r a n e s , w h i c h w e r e c l a i m e d to s h o w c y t o c b r o m e o x i d i z e a c t i v i t y . T h i s is p r o b a b l y d u e to m i t o c h o n d r i a l c o n t a m i n a t i o n . 115,116 T h e f e w t e s t e d r e a c t i o n s a r e as e x p e c t e d , b u t t h e mit o c h o n d r i a a r e n o t w e l l characterized.114-116

Mammary

Gland

T h e m i t o c h o n d r i a l s u s p e n s i o n is p r e p a r e d f r o m m o u s e o r g u i n e a pig u s i n g w e l l - w a s h e d t i s s u e o r cell s u s p e n s i o n s , in b u f f e r e d s u c r o s e - E D T A plus BSA with a loose-fitting glass-Teflon homogenizer and routine cent r i f u g a t i o n s . 117,11s Cell s u s p e n s i o n s y i e l d f e w e r b u t b e t t e r m i t o c h o n d r i a . 11s A s m u c h milk as p o s s i b l e is r e m o v e d , s i n c e this is d e l e t e r i o u s to the m i t o c b o n d r i a . 118 T h e p h y s i o l o g i c a l s t a t e o f the a n i m a l , i.e., n o n p r e g n a n t , prelactating, lactating, or postlactating, must be defined. Properties. The coupled and show velopment occurs c r e a s e in o x i d a t i v e

isolated mitochondria are morphologically intact and e n e r g i z e d ion t r a n s p o r t . 11s M a j o r m i t o c h o n d r i a l des h o r t l y a f t e r p a r t u r i t i o n in o r d e r to f a c i l i t a t e t h e inm e t a b o l i s m s e e n u p o n i n i t i a t i o n o f l a c t a t i o n . 119

Lung T h e m i t o c h o n d r i a l s u s p e n s i o n is p r e p a r e d f r o m r a t o r r a b b i t , o f t e n a f t e r in situ p e r f u s i o n w i t h saline.12°-122 T h e m i t o c h o n d r i a a r e t h e n isol a t e d in b u f f e r e d s u c r o s e p l u s E D T A 12°'121"12a'124 o r in b u f f e r e d KC1122 l14D. D. Tyler and J. Gonze, this series, Vol. 10 [17]. llSE. D. Jarasch, C. E. ReiUy, P. Comes, J. Kartenbeck, and W. W. Franke, HoppeSeyler's Z. Physiol. Chem. 354, 974 (1973). lt6 E. D. Jarasch and W. W. Franke, J. Biol. Chem. 249, 7245 (1974). 117W. L. Nelson and R. A. Butow, this series, Vol. 10 [18]. 11sC. W. Mehard, this series, Vol. 31 [65]. 119D. H. Jones and T. G. Rosano, Arch. Biochem. Biophys. 153, 130 (1972). 12°R. M. Evans and R. W. Scholtz, Biochim. Biophys. Acta 381, 278 (1975). 121R. W. Scholtz, Biochem. J. 126, 1219 (1972). IZZT. Matsubara and Y. Tochino, J. Biochem. (Tokyo) 70, 981 (1971). 123A. B. Fisher, A. Scarpa. K. F. LaNoue, D. Basselt, and J. R. Williamson, Biochemistry 12, 1438 (1973). 124H. Schiller and K. Bensch, J. Lipid Res. 12, 248 (1971).

20

ORGANELLESAND MEMaRANES

[1]

using a loose-fitting glass-Teflon homogenizer 12°-122,124 or a Polytron FF 10 tissue disintegrator, 123followed by routine centrifugations. Dithiothreitol and BSA may be beneficial, lz°,12a Potassium chloride eliminates hemoglobin adsorption to mitochondria. 122Yield: 2-3 mg protein per rat; 5-18 mg protein per rabbit. The tissue is morphologically heterogeneous so that a variety of cells will contribute to the preparation,123 and in addition contamination with lamellar inclusion bodies from alveolar cells can be expected. 122 Properties. The mitochondria oxidize citric acid cycle intermediates and glycerol 3-phosphate.12°,121,123 Bound Ca z÷ inhibits NAD-linked substrate oxidation, necessitating the presence of EDTA, but Ca z+ must be added for glycerol-phosphate dehydrogenase. 123The cytochrome content is normal.123 Pyruvate carboxylase has a very limited capacity, and pyruvate conversion to citrate is almost entirely dependent on malate.12o Earlier reports of active de novo fatty acid synthesis, believed to be involved in the synthesis of the lung alveolar surfactant dipalmitoyl lecithin, have been refuted and any de novo synthesis has been said to be of minor quantitative importance. 124There is, however, an effective fatty acid elongation system. 124

Liver (see also, this volume, Article [3]) The mitochondrial suspension is prepared from starved rats in a buffered s u cr o s e- EG T A (EDTA) medium by homogenization (glass-Teflon) or sand grinding, 125 followed by routine centrifugations, 126'127 or from isolated hepatocytes, 12s or from a tissue fractionation procedure. 6 The preparation consists of mitochondria from both parenchymal and nonparenchymal cells. These mitochondria have somewhat different properties. 129 Properties. The mitochondria oxidize intermediates of the citric acid cycle, glutamate plus malate, 3-hydroxybutyrate, la0 proline, 131 and fatty acids, all with good P/O ratios and good RCR, since phosphorylation is the limiting step. la° /3-Oxidation of fatty acids may proceed with the 125F. C. Guerra, this series, Vol. 31 [29]. lZ°L. Aringer, P. Eneroth, and L. Nordstrrm, J. Lipid Res. 17, 263 (1976). le7 D. Johnson and H. Lardy, this series, Vol. l0 [15]. 12all. Sies, T. P. M. Akerboom, and J. M. Tager, Eur. J. Biochem. 72, 301 (1977). 129T. J. C. van Berkel and J. K. Krnijt, Eur. J. Biochem. 73, 223 (1977). la0j. B. Chappell, Biochem. J. 90, 237 (1964). ~a~j. Meyer, Arch. Biochem. Biophys. 178, 387 (1977).

[1 ]

MITOCHONDRIAL PREPARATIONS AND PROPERTIES

21

formation of ketone bodies. 132 Part of the urea cycle is localized in the mitochondria, requiring a specific ornithine carrier. 133 There are two glutamate antiporters, one using hydroxyl ions and one using aspartate. 11"1°~ The amino groups for urea synthesis are derived from glutamate, 134 and the N A D P H that is required for ureogenesis from ammonia is a product of mitochondrial isocitrate dehydrogenase.128 The outer membrane may contain an aryl h y d r o c a r b o n hydroxylase that is c o m p o s e d of a cyanide-insensitive c y t o c h r o m e P-450 and that uses N A D ( P ) H as electron donor.~35 The mitochondria catalyze 26-hydroxylation of cholesterol and related compounds.~26 The mitochondria are well characterized (see, e.g., Munn136). Spleen The mitochondrial suspension is prepared from rat in buffered suc r o s e - E G T A plus BSA by glass-Teflon homogenization of a 10% mince and routine centrifugation. 75 P r o p e r t i e s . The mitochondria oxidize pyruvate plus malate, and succinate. In order to demonstrate respiratory control E G T A is essential because of the presence of an oligomycin-insensitive Mg2+-stimulated ATPase, which copurifies with the outer membrance and which continually regenerates ADP. 75

Spermatozoa The mitochondrial suspension is prepared from rabbit ejaculate or washed epidydimis 137 or from bull semen 13s by sonication 13s or by hypotonic lysis, 137followed by ccntrifugations at 900 g for l0 rain in sucrose 13s or KC1.137 P r o p e r t i e s . A sperm midpiece with the mitochondrial sheath still surrounding the axoneme, rather than free mitochondria, is isolated by this

132p. B. Garland, D. Shepherd, D. G. Nicholls, D. W. Yates, and P. A. Light, in "Citric Acid Cycle, Control and Compartmentation" (J.M. Lowenstein, ed.), p. 163. Dekker, New York, 1969. 133j. G. Gamble and A. L. Lehninger, J. Biol. Chem. 248, 610 (1973). 134S. Ratner, Adv. Enzymol. 39, 1 (1973). ~35T. Vemura and E. Chiesara, Eur. J. Biochem. 66, 293 (1976). 136E. A. Munn, "The Structure of Mitochondria." Academic Press, New York, 1974. ~37E. Keyhani and B. T. Storey, Biochim. Biophys. Acta 305, 557 (1973). ~3SH. Mohri, T. Mohri, and L. Ernster, Exp. Cell Res. 38, 217 (1965).

22

ORGANELLES AND MEMBRANES

[1]

procedure. The plasma membrane that surrounds the midpiece is removed by sonication or by hypotonic lysis, but only in the latter case do the mitochondria retain the outer membrane and lose only a small amount of cytochrome c. The mitochondria oxidize citrate, succinate, 2-0xoglutarate, pyruvate plus malate, and glycerol 3-phosphate. No NADH oxidation is found.137"138Good P/O ratios are obtained, but the presence of a flagellar ATPase causes a low RCR. 137 The high glycerol 3-phosphate respiration rate is perhaps due to the supply of glycerol from phospholipids, which are suggested to be spermatozoan substrates. The spermatozoa possess a glycerol kinase, but there is no evidence for the functioning of a glycerol phosphate shuttle.138 Neoplastic Tissue (see also, this volume, Article [9]) The mitochondrial suspension is prepared from solid tumor types, e.g., Morris hepatoma series, in buffered sucrose or mannitol-sucrose, occasionally with BSA and EDTA, by homogenization and routine centrifugations. 139,140 It is also prepared from ascites tumor cells (Ehrlich, L1210), after washing, in buffered sucrose or mannitol, occasionally containing BSA and EGTA or EDTA, by hand homogenization 141-143 or by enzymatic digestion with Nagarse 139"144 and routine centrifugations.

Properties. Mitochondria from all tumor types show unimpaired forward and reverse energy conservation and normal energy-linked activities. 139,141 There is a higher affinity for Ca 2+ than in rat liver and the presence of "superstoichiometry. ''144 No mitochondria show uncouplerstimulated ATPase. 139 Hexokinase appears to be mitochondrially bound in ascites tumor mitochondria, and glucose can therefore stimulate State 4 respiration. 142 OTHERS Insect Flight Muscle

Flies and Beetles The mitochondrial suspension is prepared from blowflies of different species (Phormia regina, Calliphora erythrocephela, Calliphora vomitora, Sarcophaga bullata, or Sareophaga nodosa Engels), from houselapp. L. Pedersen and H. P. Morris, J. Biol. Chem. 249, 3327 (1974). l*°T. M. Devlin, this series, Vo|. 10 [20]. 141R. F. W. Thorne and F. L. Bygrave, Biochem. Biophys. Res. Commun. 50, 294 (1973). 142L. A. Sauer, Biochem. Biophys. Res. Commun. 17, 294 (1964). 14SR. Wu and L. A. Sauer, this series, Vol. 10 [19]. 144B. Reynarfarje and A. L. Lehninger, Proc. Natl. Acad. Sci. U.S.A. 70, 1744 (1973).

[1]

MITOCHONDRIAL PREPARATIONS AND PROPERTIES

23

flies (Musca domestica), and from tsetse flies (Glossina moristans). It is also prepared from various beetles--Japanese beetle (Popillia japonica), Colorado beetle (Leptinotarsa decemlineata), and cockchafer (Melolontha melolontha). The isolation medium is generally lightly buffered sucrose, although KCl-based media were used previously.145-14s Recently a potassium aspartate-based medium has been successfully employed.1 Release of mitochondria is effected by digestion with Nagarse and gentle homogenization 14~'146"149 or by mechanical stirring, which also releases mitochondria with minimal damage.1 No low-speed centrifugation is required once myofibrillar components have been removed by filtration. Bovine serum albumin may be advantageous.1

Properties. The mitochondria have a very large area of inner membrane and many dense, parallel cristae.15° The membrane phospholipid composition is strikingly different from that of mammalian mitochondria (phosphatidylcholine / phosphatidylethanolamine / phosphatidylinositol 60:1 13:15 compared with 36: 40: 3). 151 There is a very high cytochrome content, respiratory activity, and phosphorylation capacity. 149,152 Since anion carriers for citric acid cycle intermediates are absent, these substrates cannot be used as exogenous substrates.1,149 No evidence exists for fatty acid or fatty acyl carnitine oxidation.147'148The only exogenous substrates that can be oxidized are glycerol 3-phosphate and pyruvate plus proline, the latter providing a condensing partner.149 Very high respiratory control ratios and rates of oxidation generally greater than 1000 nmol O per minute and milligram protein are found. Recently, rates greater than 4000 nmol O per minute and milligram protein have been observed and only these are adequate to support flight. The P/O ratios are as expected. 1The mitochondria contain high AMP levels on isolation, and AMP is formed on uncoupling, in contrast to rat liver mitochondria. This is a consequence of a dual localization of adenylate kinase in the matrix as well as in the intermembrane space. 153 Advancing senescence of the insect leads to the formation of myelinlike whorls in the mitochondria and a decrease in total respiratory chain activity, which parallels 14~R. G. Hansford and A. L. Lehninger, Biochem. J. 126, 689 (1972). 146R. G. Hansford and J. B. ChappeU, Biochem. Biophys. Res. Commun. 27, 686 (1967). ~47B. A. BuMs, S. P. Shulka, and B. Sacktor, Arch. Biochem. Biophys. 149, 461 (1972). 14sS. G. van den Bergh, this series, Vol. l0 [22]. ~4aR. G. Hansford and R. N. Johnson, Biochem. J. 148, 389 (1975). 15°D. S. Smith, J. Cell Biol. 19, ll5 (1963). 15~E. Carafoli, R. G. Hansford, B. Sacktor, and A. L. Lehninger, J. Biol. Chem. 246, 964 (1971). 152H. Wohlrab, Biochemistry 13, 4014 (1974). ~53S. M. Danks and J. B. Chappell, Biochem. J. 142, 353 (1974).

24

ORGANELLES AND MEMBRANES

[1]

a d e c r e a s e d ability to sustain flight. 147,1,54As in m a m m a l i a n mitochondria, glycerol-3-phosphate d e h y d r o g e n a s e is stimulated by Ca 2+. 146,155 Massive and rapid m o n o v a l e n t cation p e r m e a t i o n is electrophoretic and requires a potential gradient and p e r m e a n t anions. 145 Calcium ion transport is unclear. An earlier report indicated no high-affinity sites and v e r y few of low affinity. 151 Recently, there has b e e n a d e m o n s t r a t i o n o f an energylinked Ruthenium-red-sensitive Ca 2+ transport with less than one carrier p e r c y t o c h r o m e oxidase. 152 N A D - L i n k e d isocitrate d e h y d r o g e n a s e is under adenine nucleotide control and is believed to be the p r i m a r y control locus o f the citric acid cycle. 149,155,156

Locust The m i t o c h o n d r i a are isolated in a sucrose-based or K C l - b a s e d medium 14s'15r b y digestion with N a g a r s e and gentle homogenization. 157

Properties. In contrast to blowfly, these mitochondria p o s s e s s citric acid cycle carriers and can therefore oxidize glutamate plus malate and also fatty acyl carnitine plus malate. 14s Glycerol-3-phosphate dehydrogenase activity is high, and the e n z y m e is localized on the outer surface of the inner m e m b r a n e . 15s It is involved in the " g l y c e r o l p h o s p h a t e shutt l e " for oxidation of c y t o p l a s m i c NADH.lS7 Higher Plants The mitochondrial suspension is p r e p a r e d f r o m etiolated mung b e a n h y p o c o t y l (Phaseolus aureus), potato tuber (Solanum tuberosum), Jerusalem artichoke (Helianthus tuberosus), cauliflower (Brassica oleracea var. botrytis), skunk, cabbage spadix (Symplocarpus foetidus), sweet potato (Ipomea batatas), turnip (Brassica rapa), and others. The main p r o b l e m s are contamination with other organelles ( p e r o x i s o m e s , glyoxy s o m e s , and chloroplasts) and release of phenolics and quinones from the central vacuole. M E R C A P (2-mercaptobenzothiazole) or P V P (polyvinylpyrrolidone) is added to o v e r c o m e the latter. The tissue can be h o m o g e n i z e d in m a n y different ways in a buffered sucrose or mannitol m e d i u m (plus E D T A , BSA, glutathione, or cysteine). ( F o r a general description of preparations, see Laties. 159) The mitochondria must be 154B. A. Bulos, S. P. Shulka, and B. Sacktor, Arch. Biochem. Biophys. 166, 639 (1975). 155R. G. Hansford and B. Sacktor, J. Biol. Chem. 245, 991 (1970). 15hR. N. Johnson and R. G. Hansford, Biochem. J. 146, 527 (1975). 157W. J. Lloyd and R. Harrison, Arch. Biochem. Biophys. 163, 185 (1974). 1~8M. Klingenberg, Eur. J. Biochem. 13, 247 (1970). I59G. G. Laties, this series, Vol. 31 [62].

[1]

MITOCHONDRIAL PREPARATIONS AND PROPERTIES

25

purified on a discontinuous sucrose density gradient in order to show acceptable RCR and respiratory rates. 160

Properties. The mitochondria oxidize citric acid cycle intermediates and N A D H . Upon successive additions of ADP, the State 4 respiration becomes slower (termed "conditioning").16°-162 The mitochondria contain more flavoproteins than do animal mitochondria, and cytochromes b-553, b-557, b-562 (perhaps not in the respiratory chain), c-549, c-547, a, and aa. 16a They may lack N A D P H . 164 There are two pathways for N A D H oxidation: the usual internal one and one on the outer membrane, a rotenone-insensitive N A D H reductase ( N A D P H cannot serve as an electron donor), from which electron transport is coupled only at sites II and III. 16~,166Malate can be oxidized both through malate dehydrogenase and through the NAD-requiring malic e n z y m e to yield pyruvate.16~ The succinyl-CoA synthetase uses ADP, not GDP as in animals. 16r Upon aerobic incubation of storage tissue, a mitochondrial cyanide-insensitive, nonphosphorylating electron-transport chain is developed. It appears to be inoperative in vivo 163 but may be of importance in skunk cabbage spadices, which are thermogenic.168'169 It is inhibited by aromatic hydroxamic acids.168'169 The mitochondria possess a sulfite oxidation system 17° and ferrochelatase. 171 The length of the mitochondrial D N A is 35 /~m. 12

N e u r o s p o r a crassa (see also, this v o l u m e , Article [18]) The main difficulty in mitochondrial isolation is the hard heteropolysaccharide cell wall. In the past, this was broken by glass beads (poor RCR) or by snail gut e n z y m e 17z or by pure fl-glucuronidase.173 These methods give either mitochondria of poor quality or a low yield. Grinding 16°R. Douce, E. L. Christensen, and W. D. Bonner, Biochim. Biophys. Acta 275, 148 (1972). ln~j. K. Raison, J. M. Lyon, and L. C. Campbell, J. Bioenerg. 4, 397 (1973). ~62j. K. Raison, G. G. Laties, and M. Crompton, J. Bioenerg. 4, 408 (1973). 163S. Yoshida, Annu. Rev. Plant Physiol. 23, 437 (1972). 1n4H. Ikuma, Science 158, 529 (1967). ~65j. D. Coleman and J. M. Palmer, Eur. J. Biochem. 26, 499 (1972). 166D. A. Day and J. T. Wishich, Arch. Biochem. Biophys. 171, 117 (1975). 167j. M. Palmer and R. T. Wedding, Biochim. Biophys. Acta 113, 167 (1966). ~ngj. T. Bahr and W. D. Bonner, J. Biol. Chem. 248, 3441 (1973). ~69j. T. Bahr and W. D. Bonnet, J. Biol. Chem. 248, 3446 (1973). 170j. M. Tager and N. Rautanen, Biochim. Biophys. Acta 18, I l l (1955). t7~H. N. Little and O. T. G. Jones, Biochem. J. 156, 309 (1976). a7zj. W. Greenawalt, D. O. Hall, and O. C. Wallis, this series, Vol. l0 [27]. ~TaD. L. Edwards and E. Rosenberg, Eur. J. Biochem. 62, 217 (1976).

26

ORGANELLES AND MEMBRANES

[1]

in a special mill in 440 m M sucrose ( E D T A , BSA) is an acceptable c o m p r o m i s e . 174 F u r t h e r preparation is carried out by routine methods. The mitochondrial p r e p a r a t i o n shows l y s o p h o s p h o l i p a s e activity, which can be r e m o v e d on a sucrose density gradient.175 P r o p e r t i e s . The mitochondria oxidize p y r u v a t e plus malate, and succinate, but not 2-oxoglutarate, ethanol, or lactate when a s s a y e d in a 440 m M s u c r o s e - b a s e d incubation medium. T h e y also oxidize N A D ( P ) H and contain b o t h the usual cristae m e m b r a n e N A D H d e h y d r o g e n a s e (rotenone sensitive P/O = 3) and an " o u t e r " N A D ( P ) H d e h y d r o g e n a s e (rotenone insensitive, P/O = 2). The c y t o c h r o m e s are (liquid-nitrogen spectrum) as follows: a-605, b-563, b-556 (not found in yeast), c-548, and c538 (as in yeast). In N e u r o s p o r a in the logarithmic phase the a/b/c ratio is 1 : 2:4.174 S o m e mutants (e.g., cni-1) have a cyanide-insensitive respiration, which also can be induced by chloramphenicol inhibition of mitochondrial protein synthesis in the wild type. This cyanide-insensitive respiration is not mediated through c y t o c h r o m e s 176 and, once induced, c y t o p l a s m i c protein synthesis is n e c e s s a r y to o v e r c o m e it. it3 The mitochondria are used mainly for investigations of mitochondrial protein synthesis (hydrophobic c o m p o u n d s of c y t o c h r o m e oxidase, c y t o c h r o m e b, and A T P synthetase).177 A p r e c u r s o r of c y t o c h r o m e oxidase has b e e n identified. 178,179 In cni-1, which lacks the normal mitochondrially synthesized peptides, only a peptide, H P 8500, is synthesized.177 The length of the mitochondrial D N A is 20 ftm.12

Y e a s t (see also, this v o l u m e , Articles [19] a n d [20]) The mitochondrial suspension is p r e p a r e d from S a c c h a r o m y c e s c e r e visiae, S a c c h a r o m y c e s c a r l b e r g e n s i s , and C a n d i d a utilis ( T o r u l o p s i s utilis). M a n y mutants are used in genetic studies. The cells are harvested in the selected growth stage, well washed in distilled water, and resuspended in sorbitol (0.6-1.3 M) containing E D T A and a buffer, p H 5.86.4. T h e y are then treated sequentially or simultaneously with a sulfhydryl reagent ( m e r c a p t o e t h a n o l , cystamine) and snail gut e n z y m e at 30 ° 174H. Weiss, G. von Jagow, M. Klingenberg, and T. Bficher, Eur. J. Biochem. 14, 75 (1970). 175G. W. de Goede, J. Samallo, M. Haltrop, and G. L. Scherphof, Biochim. Biophys. Acta 424, 195 (1976). lrrD. L. Edwards, E. Rosenberg, and P. A. Maroney, J. Biol. Chem. 249, 3551 (1974). 177R. Michel, A. Liebl, W. Machleidt, J. Otto, and W. Neupert, Hoppe-Seyler's Z. Physiol. Chem. 356, 1595 (1975). trsS. Werner, Eur. J. Biochem. 43, 39 (1974). 1795. Werner, A. J. Schwab, and W. Neupert, Eur. J. Biochem. 49, 607 (1974).

[1 ]

MITOCHONDRIAL PREPARATIONS AND PROPERTIES

27

for 30-90 min until protoplasts are formed. The protoplasts are lysed by osmotic shock (and gentle homogenization) and the mitochondria collected by routine centrifugations, ls0-,a~ The yield of mitochondria is 2.5 mg protein per gram wet washed cells. An alternative procedure, entirely mechanical, involves a colloid mill or homogenizing with glass beads. The mitochondria are released directly and collected as usual, la6,,s7 The quality is reproducible, but the yield is only 13-25 mg protein per 100 g packed cells.

Properties. The mitochondria are very stable, giving unchanged P/O ratios between pH 5.4 and 7.9 and between 0.3 and 1 M tonicity. Tonicities used are often about 0.6 M. 182,183,185Saccharomyces and Candida oxidize citric acid cycle intermediates, glycerol 3-phosphate, lactate, and ethanol. ,SZ,la3,1as,,s6The latter two are unusual mitochondrial dehydrogenases.'85 A carrier for pyruvate has been reported which also transports lactate, lS8 Under normal growth conditions Candida oxidizes NAD-linked substrates with a P/O ratio approaching 3. H o w e v e r , during iron or sulfur deficiency, the site I phosphorylation and rotenone sensitivity disappear. An external rotenone-insensitive N A D H dehydrogenase appears on the outer surface of the inner membrane.lS~,lsg,'9° In Saccharomyces under most isolation conditions site I phosphorylation is lacking, and P/O ratios of 2 are obtained with NAD-linked substrates. Then N A D H is also oxidized at a high rate.'82-186,'9° This resembles the situation induced in Candida. Saccharomyces does, however, appear to be genetically competent in site I. High-affinity Ca z+ binding is lacking, although at very high concentrations some respiration-driven Ca 2+ uptake can be obtained. 191,192 Since the cytochromes are inducible depending on oxygen ~s0j. B. Chappell and R. G. Hansford, in -Subcellular Components. Preparation and Fractionation" (G. D. Birnie, ed.), 2nd ed., p. 77. Butterworth, London, 1972. ,s,j. C. Beck, J. R. Mattoon, D. C. Hawthorne, and F. Sherman, Proc. Natl. Acad. Sci. U.S.A. 60, 186 (1968). tS2M. Briquet, N. Sabadie-Pialoux, and A. Goffeau, Arch. Biochem. Biophys. 174, 684 (1976). 'S3T. Ohnishi, K. Kawaguchi, and B. Hagihara, J. Biol. Chem. 241, 1797 (1966). lS4L. Kov~iS, H. Bedn~irovfi, and M. Greks~ik, Biochim. Biophys. Acta 153, 32 (1968). 185G. Jagow and M. Klingenberg, Eur. J. Biochem. 12, 583 (1970). '86W. X. Balcavage and J. R. Mattoon, Biochim. Biophys. Acts 153, 521 (1968). as7 M. Somlo and M. Krupa, Eur. J. Biochem. 42, 429 (1974). 'SSM. Briquet, Biochim. Biophys. Acta 459, 290 (1977). ,89p. B. Garland, Biochem. J. 118, 329 (1970). '9°T. Ohnishi, Biochim. Biophys. Acta 301, 105 (1973). 19~W. X. Balcavage, J. L. Lloyd, J. R. Mattoon, T. Ohnishi, and A. Scarpa, Biochim. Biophys. Acta 305, 41 (1973). ,92 E. Carafoli, W. X. Balcavage, A. L. Lehninger, and J. R. Mattoon, Biochim. Biophys. Acta 205, 18 (1970).

28

ORGANELLES AND MEMBRANES

,t,~J

supply, energy source, and stage of development, mitochondrial function and development are amenable to analysis in yeast.18~'18"i"193Yeast mitochondrial DNA is 25 /zm ~2 and has been used for genetic mapping and studies of mitochondrial protein synthesis. Promitochondria 193

Promitochondria are isolated from anaerobic S. cerevisiae and lack cytochromes aa3, b, cl and c and ubiquinone. They have a low ergosterol content and low degree of fatty acid unsaturation. Upon oxygenation they are converted to respiring mitochondria. They can be isolated by the same procedure as that for aerobic mitochondria except that cycloheximide must be present, and the promitochondria are more fragile. They should therefore not be purified on a sucrose gradient. 19aG. Schatz and I. Kov(t~, this series, Vol. 31 [65].

[2] L o n g - T e r m S t o r a g e o f M i t o c h o n d r i a t o P r e s e r v e Energy-Linked Functions 1 By

SIDNEY FLEISCHER

In this article a procedure is described for the storage of rat liver mitochondria in order to preserve mitochondrial functions for prolonged periods of time (months). The availability of many tubes of mitochondria from one large-scale preparation emancipates the investigator from the daily tedium of isolation. Such a stock eliminates individual differences in animals and inadvertent changes in the isolation procedure. Three parameters of mitochondrial function were tested (phyosphorylation efficiency, respiratory control, and Mg 2+ control of energized swelling), and each was preserved by this method. Principle Mitochondria are fortified with bovine plasma albumin and dimethyl sulfoxide (10%, v/v), a cryoprotective substance. They are quick-frozen in liquid nitrogen and stored in a liquid-nitrogen refrigerator at -196 °. 2 Supported in part by Grant AM 14632 from the National Institutes of Health. I am grateful to Dr. J. Oliver McIntyre for his c o m m e n t s on the manuscript. 2 K. G. Walton, M. Kervina, S. Fleischer, and D. S. Dow, Bioenergetics 1, 3 (1970). METHODS IN ENZYMOLOGY,VOL. LV

Copyright © 1979by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181955-8