Preservation of native properties of mitochondria in rat liver homogenate

Mitochondrion 1 (2001) 249–267 www.elsevier.com/locate/mito

Preservation of native properties of mitochondria in rat liver homogenate M.N. Kondrashova*, N.I. Fedotcheva, I.R. Saakyan, T.V. Sirota, K.G. Lyamzaev, M.V. Kulikova, A.V. Temnov Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, 142290, Russia Received 26 March 2001; received in revised form 29 June 2001; accepted 9 July 2001

Abstract A protocol is developed for preparation of concentrated rat liver homogenate preserving assemblies of mitochondria in isotonic KCl under 0 and 158C. Assemblies preserve ability for self-organization during storage in homogenate. All key energy functions of mitochondria can be investigated in such a homogenate. Oxidative phosphorylation and membrane potential are stable for 5–7 h and can be still observed on the next day. Substrate-level phosphorylation is better pronounced for mitochondria in KCl than in sucrose medium while Ca 21 capacity is greater and lipid peroxidation is much lower. Sucrose addition impairs these functions. The rate of phosphorylating respiration is lower in large assemblies and higher in small. Transition from large to small assemblies corresponds to the transition from quiescent state of animal to adrenaline induced active state. The proposed method is particularly convenient for clinical investigations with small bioptates. q 2001 Elsevier Science B.V. and Mitochondria Research Society All rights reserved. Keywords: Mitochondria; Mitochondrial network; Respiration; Guanidine triphosphate; Adrenaline

1. Introduction

When you purify an enzyme, make sure it stays alive within its biological community (Racker, 1976) The study of mitochondria is growing in importance. Many physiological and pathological states of the organism have been shown to depend on changes in metabolic processes in the mitochondria. Regulation of mitochondrial processes emerges as a new approach to correcting pathology. Two crucial steps * Corresponding author. Tel./fax: 17-0967-79-05-53. E-mail address: [email protected] (M.N. Kondrashova).

in the development of physiological study of mitochondria should be remembered. The first was the discovery of metabolic states of mitochondria by Chance, in particular of State 4, controlled, or quiescent (Chance and Williams, 1955). This state made it possible first to reproduce in vitro essential physiological ability of living tissues, in vivo to reverse their response to energy load with its compensation and return to initial level, and even higher (Kondrashova, 1973). This phenomenon demonstrated the energy control of respiration and led to the understanding of its role in maintaining normal physiological state. The second step was the discovery of the Luft disease showing that loss of energy control of respiration is inherent to pathology, particularly non-thyroid hypermetabolism related to muscle fatigue (Ernster et

1567-7249/01/$20.00 q 2001 Elsevier Science B.V. and Mitochondria Research Society All rights reserved. PII: S 1567-724 9(01)00025-3

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al., 1959). This phenomenon revealed the role of the impairment of mitochondria in the development of diseases. These discoveries provided the basis for physiological investigations of mitochondrial processes. Now physiological aspects of mitochondriology are developed mostly in the context of studying mitochondrial diseases and apoptosis. The same protocols for isolation of mitochondria, which were developed several decades ago for obtaining ‘pure’ mitochondrial fraction free from other cytoplasmic components, are still used in modern physiological investigations. These protocols made it possible to find out what processes occur in the mitochondria. However, separation of mitochondria from their natural environment can disturb some of their native properties. The most obvious example of this is the loss of the inherent property of mitochondria to form assemblies. This property is so basic that it gave mitochondria their name: mitochondrion – thread of beads. Modern electron microscopic data demonstrated the existence of mitochondrial network within the cell, in which the mitochondria are tightly bound to the reticulum. However, these bindings must be destroyed for the isolation of mitochondria for the organelles to be obtained as separate granules. Nonpolar sucrose diminishes structural interactions not only between mitochondria but also between their components because the matrix as well as the membrane are ordered. Sucrose decreases the matrix volume while KCl increases it (Devin et al., 1996, 1997a,b; Garlid, 1994). Ca 21-dependent K 1 induced swelling of mitochondria provides stimulation of respiration by adrenaline, its agonists, and synergists (Siess and Wieland, 1980; Halestrap, 1989; Halestrap et al., 1986, 1990; McCormack and Denton, 1990). Changes in the matrix volume serves as the mechanism of regulation of mitochondrial functions (Haussinger et al., 1990; Haussinger and Lang, 1991; Baquet et al., 1990; Meijer et al., 1992). Not much is known about how disassembling mitochondrial network influences the functions of mitochondria. Matrix enzymes and oxidative phosphorylation are more active in fractions of heavy mitochondria consisting of fragments of mitochondrial– reticular chain (Katz et al., 1983). Traditional cooling during isolation destroys filamentous organization of mitochondria and abolishes in vitro, the differences

exhibited in vivo, while warm isolation under 158C preserves them (Gosalvez et al., 1996). We developed a method for preserving assemblies of mitochondria in concentrated isotonic KCl homogenate and investigated the dependence of their energy function on the size of assemblies as measured by video microscopy (Kondrashova et al., 1997; Temnov et al., 2000a,b,c). We found that respiration is higher in smaller assemblies. Reversible assembling of mitochondria serves as the mechanism for control of respiration. Addition of sucrose to KCl decreases respiration considerably. Because isotonic KCl is close to natural cytosol medium and native assemblies of mitochondria are preserved in isotonic KCl, decrease of respiration in sucrose medium should probably be considered as an artifact. These results agree with other data (Chavez et al., 1997; Devin et al., 1997a) and with the conclusion: ‘when isolated mitochondria are studied in KCl medium, matrix volume and oxidative phosphorylation activity tend to be regulated as in cells’ (Devin et al., 1997a). Besides the presence of sucrose, other standard conditions of isolation, such as high dilution of tissue and traditional cold also destroy native assembling of mitochondria (Hansford, 1978; Kondrashova et al., 1997; Temnov et al., 2000b). The impairment of native state is particularly undesirable in physiological investigations of mitochondria since it can eliminate physiological regulation of mitochondrial processes. Fortunately, since localization of energy processes within the mitochondria is now established, there is no need to isolate pure mitochondria in all investigations. Breaking native connections of mitochondria with the reticulum, putting them in a medium with lowered protein content, and exposing them to disassembling conditions are not necessary for studying energy functions of mitochondria. Mitochondria can be studied in concentrated isotonic KCl homogenate, rather than being separated from their biological environment. In assemblies, mitochondria not only retain their native structure but apparently ‘stay alive’ (see epigraph) since assemblies can grow after isolation. Using homogenate considerably simplifies and shortens the whole preparation procedure. Biochemical measurements can be started in less than 10 min after taking a tissue. Another advantage of using a homogenate is that in the medium of native composition, lipids of mito-

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chondrial membrane are less oxidized than in standard preparations and energy functions are stable for several hours and are preserved even the next day. Preparation stability is of great importance for clinical investigations of bioptates, which are sometimes delivered to the laboratory several hours after being taken from a patient. One more important advantage of concentrated homogenate is that it contains the whole population of mitochondria in tissue. Under standard isolation, a portion of heavy mitochondria is lost with the first pellet and the fluffy layer (swollen mitochondria) is discarded. Swollen mitochondria probably correspond to the activated fraction. Low dilution of tissue and isotonic KCl provide a real opportunity to prepare unimpaired mitochondria from a small piece of tissue. In this paper, we describe improvements to the method of obtaining assemblies of mitochondria in concentrated isotonic KCl homogenate which keep oxidative phosphorylation stable for at least 5–7 h and preserve it even until the next day. We also report data on substrate-level phosphorylation (SP), membrane potential, Ca 21 capacity, and other properties of mitochondria in the homogenate. 2. Materials and methods

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7.5 and additions for energy processes given in figure captions. Sucrose medium for homogenization (and storage) contained 250 mM sucrose instead of KCl, other additions as in KCl medium. Sucrose–KCl medium for respiration contained 150 mM sucrose and 50 mM KCl, other additions as in KCl medium for incubation. Preparation procedure is rapid. After decapitation, the liver was cut, rinsed with homogenization medium (instead of perfusion used before), crushed through iron steel press, and gently homogenized manually in a loose Teflon-glass homogenizer by five to six strokes with the addition of 1 ml medium per 1 g of tissue (final concentration 80–100 mg protein/ml). Before the tissue is destroyed, its temperature for cold or warm preparations should reach the necessary temperatures of homogenization, 0 or 158C, respectively. This demands 2–4 min rinsing and keeping in solution in the given temperature. For correct cold storage, the homogenate should be kept in closed small vial and inserted deeply into a plastic bottle filled up with ice and additionally kept in the refrigerator between sample taking. If the homogenate is not well protected from room temperature its inside temperature can be close to 4–58C, which is damaging. Some details of preparation are also described elsewhere (Kondrashova et al., 1997).

2.1. Animals and animal treatment

2.3. Isolation of mitochondria

Male Wistar rats weighing 200–220 g bred in an ITEB vivarium were used throughout the work. Animal treatment provided their quiescent state. Adrenaline (25 mg/100 g body weight) was administered intraperitoneally to the experimental animal 30 min before decapitation. In order to maintain control animals intact, they were not injected with saline.

For comparison with the homogenate, mitochondria were isolated from the other half of the same liver. Homogenization medium contained 0.30 M sucrose, 10 mM Tris, pH 7.5, tissue/medium ratio: 1/8. The first centrifugation was done at 1700 £ g for 7 min and the second centrifugation at 5500 £ g for 20 min. It was determined that the concentration of mitochondrial protein in the homogenate is 25% of the total protein.

2.2. Preparation of assembled mitochondria 2.4. Video microscopy KCl medium for homogenization (and the following storage) contained 125 mM KCl, 10 mM HEPES, pH 7.5 (instead of 7.2 previously used). EGTA, 1 mM, can be added in pure in vitro experiments but it should be omitted in investigations of responses induced in vivo as this eliminates Ca 21-mediated hormone activation. KCl medium for incubation is similar to homogenization, EGTA omitted, pH 7.2 instead of

Assemblies of mitochondria were observed by light microscopy in dark field or phase contrast without fixation or after staining with nitro-blue tetrazolium. For staining, smear of homogenate was made which rapidly dried in air, then fixed by cold (28C) acetone for 30 s, and incubated in KCl medium with succinate and dye for 60 min at 378C.

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Video camera MINTRON CCD MTV-1 802 CB combined with microscope MBI-11 was used for observations. The velocity of video catch is about three sequences per second. The video camera was attached to the PC Pentium 133 with video card coupled device for image entry DE-12B2 from ‘Kandela’ Company, Moscow, Russia. Micro-objects were detected and calculated using UTHCSA Image Tool, version 2.00 software. The level of scanning was selected on the basis of automatic determination of background density and gray level mode (256 total). The level of scanning was mode 1 45. For microscopic measurements, 30 ml of homogenate was added in 1 ml of homogenization medium, stirred with low speed stirrer for 5 min, and observed. These procedures were carried out at 16–188C. Dilution and stirring are necessary for morphometry because initial homogenate is too thick and after addition of solution it should be mixed to provide a homogeneous suspension. However, stirring destroys the assemblies. A 5 min stirring provides a completely homogeneous suspension. In spite of some disassembling (the number of assemblies is increased per 10– 30%) this point is convenient for comparisons. It should be noted that in the incubation medium with substrate, phosphate and formed adenosine triphosphate (ATP), the assemblies are less stable. After 4–5 min of respiration measurement, only 20– 30% of mitochondria are assembled and these assemblies are small. This will be discussed. Results are presented as the mean area of assemblies. Each point on the morphometric graphs is the mean of scanning nine fields of vision containing total number of 800–2000 objects. 2.5. Biochemical methods The measurements of energy functions were carried out at 268C. Polarographic curves were transformed into diagrams by computer program of A.N. Fedyanin. Respiration was measured by polarographic method (Chance and Williams, 1955). SP was registered as self-inhibition of mild uncoupled a-ketoglutarate oxidation (KGL) (Olson and Allgyer, 1973a). Homogenate (50 ml) in 1 ml of medium was used for respiration measurements. Membrane potential was measured with TPP-electrode (Kamo et al.,

1979). Homogenate (20 ml) in 1 ml of medium was used in membrane potential measurements. Ca 21 capacity was measured either with Ca 21 or H 1 electrodes. The total accumulation of Ca 21 was determined by addition of small portions. Concentration of protein was as in respiration measurements. Protein was measured according to Lowry et al. (1951) and lipid peroxides were assayed by Ohkava et al. (1979). Each point of respiration data is the result of one to two measurements with the accuracy of 1–3%. Accuracy of membrane potential and Ca transport measurements are similar those of respiration. Statistical treatment was made by Student method and pair criterion pT (Bailey, 1963). 2.6. Chemicals HEPES, EGTA, succinate, and adenosine diphosphate (ADP) sucrose (ultra) were from Sigma (St. Louis, MO, USA). Potassium salts chemically pure or highly pure were from Reakhim (Moscow, Russia). 3. Results Our procedure has three essential differences from standard isolation of mitochondria: KCl is used instead of sucrose, low dilution of tissue instead of high, and temperature of 158C instead 08C in one variant. 3.1. Video microscopy of homogenate. Selfassembling of mitochondria in vitro Two samples of mitochondria in homogenate are presented in Fig. 1. In contrast to standard preparations, mitochondria are gathered in assemblies with different mean area 10–20 mm 2 in the upper panel and 30–60 mm 2 in the lower. Nuclei which are well seen in the homogenate can serve as a scale, with a diameter of about 10 mm 2. More fragile than the mitochondria, the nuclei are well preserved during the whole day storage. This provides evidence that the medium is not damaging. In order to test assemblies as mitochondria they were vitally stained with rhodamine or yanus-green (data not shown). Both the dyes were accumulated in assemblies. In the presence of 2,4-dinitrophenol (DNP) or dicumarol only very weak non-specific staining was developed and the assem-

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of native mitochondria in the cell. They are seen in the smear of homogenate stained with nitro-blue teterazolium in the presence of succinate. This detects succinate dehydrogenase, mitochondrial marker. These filamented mitochondrial structures are presented in Fig. 3. Like in the cell, granules (beads) are seen situated along filamentous structures and separated with small spaces. These data provide evidence that the mitochondria in assemblies retain the native filamentous structure. Fig. 4 demonstrates that sucrose and cold induce dissociation of assemblies, while KCl and warmth maintain the mitochondria assembly. It is also shown that the assembled state of mitochondria is

Fig. 1. Assemblies of mitochondria in concentrated rat liver homogenate stored in cold. Vital phase-contrast microscopy, magnification £116. Rinsed from blood, then minced liver was gently, manually homogenized in 125 mM KCl, 10 mM HEPES, pH 7.5 and added was 1 ml per 1 g tissue (80–100 mg protien/ml) in icecold conditions. Upper panel – freshly prepared homogenate, lower panel – in 1 h storage in ice. Assemblies of mitochondria and wellpreserved nuclei are seen. Nucleus diameter of about 10 mm can serve as a visual scale of size. Enlargement of assemblies occurs during storage.

blies were dissociated. This provides evidence that the assemblies are formed by energized mitochondria. In the course of storage, the size of the assemblies is increased. Under isolation and storage in cold, enlargement is slow and small. The enlargement is greater when the homogenate is prepared in warm temperature (158C). In this case, the initial size is smaller than in cold but this is considerably increased during storage also in warmth as shown in Fig. 2 Assemblies grow due to the joining small ones to a larger one. After storage in warmth, the small assemblies disappeared. Large assemblies contain ‘thread of beads’ typical

Fig. 2. Assemblies of mitochondria in concentrated rat liver homogenate stored in warm condition. Vital dark-field microscopy. Homogenate was prepared as in Fig. 1, but under 158C. Upper panel – freshly prepared homogenate, lower panel – in 1 h storage at 16–188C. Pronounced enlargement of assemblies occurs during warm storage. Well-preserved nuclei are seen in both cases.

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medium for 5 min reveals different reserve of stability of assemblies stored in cold or warmth. Transfer of the cold homogenate results in a near two-fold decrease of area in both control and adrenaline administered animals, although their initial size was different – large and small, respectively. Transfer of the warm homogenate of control animal to warm condition decreases the area of the initially largest assemblies to only per several percents. The decrease of area in the warm homogenate of adrenaline-administered animal is only 15% instead of 60% in cold homogenate. The decrease of absolute value of area of assem-

Fig. 3. Filamentous mitochondria in a smear of rat liver homogenate. Dark-field microscopy. Homogenate preparation as in Fig. 1. Thin smear on a glass was prepared from a drop of homogenate, rapidly dried in air, fixed for 30 s in ice-cold acetone and stained with nitro-blue tetrazolium in the presence of succinate for succinate dehydrogenase. Filamentous mitochondrial structures, threads of beads, typical of native cell are clearly seen.

typical of quiescent state of animals: assemblies are greater in intact animals and dissipate after injection of adrenaline to the animal. Homogenates were prepared and stored for about 1 h in the KCl medium under cold and warm conditions. Two halves of the same liver were used for comparison. Changes in the mean area of assemblies were measured in the course of storage as shown in Fig. 4. The last point of each series presents data for sample transferred from KCl to sucrose–KCl medium under 16–188C and stirred for 5 min before the area of assemblies was measured. Assemblies from control animals are greater than after adrenaline administration. The initial mean area is greater under preparation in cold than in warmth. However, further assembling is considerably greater during storage in warmth than in the cold. Under warm storage, the mean area reached 47 mm 2 instead of 37 mm 2 under cold condition. The relative increase in warmth is 175% of initial area while this is only 15% for cold storage. As can be seen, the area of assemblies is smaller after adrenaline administration under both cold and warm conditions. The relative differences between control and adrenaline administered animals in KCl are similar in cold and warm pairs and are about 250% in 1 h. Transfer from KCl to sucrose–KCl

Fig. 4. Time course of self-assembling of mitochondria during storage under different conditions. Pair experiment was performed in two halves of the same liver. Homogenate was prepared as in Fig. 1 but one portion under ice-cold conditions and the other at 158C. Then, homogenates were stored under two respective temperatures of preparation. The samples for dark-field microscopy and morphometry were taken in the course of storage. Triangles, warm preparation and warm storage; squares, cold preparation and cold storage; open symbols, control animal; dark symbols, adrenaline administered animal. The last point under each curve presents data for a sample of KCl homogenate transferred into sucrose–KCl medium for 5 min, 16–188C. Dissipation of assemblies is a measure of their stability. Data demonstrate that assembling is facilitated in vitro by KCl and warmth, and in vivo by the quiescent state of animal. Disassembling is induced in vitro by sucrose and cold, and in vivo by adrenaline.

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blies in adrenaline administered animal compared with control is similar: 20–23 mm 2 in three variants, in KCl medium in warm and cold homogenate and in sucrose–KCl medium in warm homogenate. However, this is considerably less, 8 mm 2 in sucrose–KCl medium and cold homogenate. This also provides evidence that sucrose and cold are synergists in disassembling. The lesser stability of assemblies formed in the cold suggested that a part of the mechanisms causing assembling in warm conditions does not work in the cold. The rate of self-assembling can also serve as a convenient test which helps to compare samples with different kinetics. The rate of increase in size can be calculated for different time intervals. For example, in the warm homogenate this is 0.31 mm 2/ min for control and 0.18 mm 2/min for adrenaline measured in 10–70 min incubation. Fig. 4 demonstrates the main results of assembling dependence on the experimental conditions: KCl and warm condition favor preservation of assemblies and to their self-organization. In contrast, cold and sucrose facilitate disassembling. Assemblies and their selforganization are greater in intact animal and smaller under adrenaline administration.

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3.2. Phosphorylating respiration of assembled mitochondria Phosphorylating respiration of assembled mitochondria in homogenates was found to be very stable during storage. The typical examples of polarographic registration of respiration of mitochondria in freshly prepared homogenate, 1 h after isolation and on the next day are given in Fig. 5. As shown Chance’s respiratory control is well pronounced in the homogenate. Parameters of respiration are not impaired during 3–6 h storage. Respiratory control is even observed when measured next day. For convenience of comparison, polarographic curves were transformed into diagrams. Further diagrams of respiration will be presented. 3.3. Protocol of in vitro examination of the state of mitochondria in vivo: simultaneous measurements of respiration and area of assemblies Fig. 6 shows diagrams of respiration in the same homogenates in which the area of assemblies was measured under different conditions and presented in Fig. 4. Homogenates were obtained from control

Fig. 5. Phosphorylating respiration of mitochondria in the rat liver homogenate during prolonged storage. KCl homogenate, cold prepared and cold stored as in Fig. 1. 1, freshly prepared; 2, 1 h storage; 3, 24 h storage. First arrow at each curve – ADP addition, second arrow – DNP addition. The end of curve 3 shows zero level of oxygen. Basal incubation medium – 125 mM KCl, 10 mM HEPES, pH 7.2. Additions – 2 mM succinate, 1 mM KH2PO4, and 200 mM ADP. After phosphorylation, 5 £ 10 25 M 2,4-DNP was added. Temperature of incubation is 268C. Here and in all biochemical measurements, the homogenate was taken by pipette with wide tip: obliquely cut top of an automatic pipette. Protein concentration in the incubation medium is 4–5 mg/ml. Stirring in the cell was as slow as possible.

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Fig. 6. Phosphorylating respiration of mitochondria in the rat liver homogenate under different conditions. The simultaneous experiment to morphometric measurements are given in Fig. 4. Conditions are described in the legend for Fig. 4. Two halves of the same liver were used for comparison. Panels A and B: KCl homogenate, I, warm prepared and stored, II, cold prepared and stored. Panel A, incubation in KCl medium; panel B, incubation in sucrose–KCl medium; thin lines, control animal; thick line, adrenaline administered animal. The first two pairs in each panel – freshly prepared homogenate, the second two pairs in panel A in 2 h storage in cold and warmth. Percents show adrenaline stimulation of control.

and adrenaline administered animals in KCl medium in cold and warm conditions, two halves of the same liver were used for comparison. Homogenates were stored under the temperature of preparation and then incubated under 268C in KCl or sucrose–KCl. The main results of this experiment are as follows. (1) Respiration of warm homogenate is moderately lower than that of cold. This difference increases in 2 h. (2) Respiration and energy control in homogenate are not declined during at least 2–6 h storage. During storage, a moderate increase of State 3 respiration is found for cold homogenate and a small decrease for the warm one in KCl medium of incubation. The decrease in rate of the warm homogenate was coupled with the prolongation of phosphorylation duration, which was not changed in the cold homogenate. (3)

Respiration in the sucrose–KCl medium is considerably lower for both, warm and cold homogenate and the duration of phosphorylation is prolonged compared with the KCl incubation medium. (4) In all cases, respiration of adrenaline administered animal is higher than control. Increase in percent is the highest in freshly prepared warm homogenate and in cold homogenate after storage in both KCl and sucrose–KCl media. (5) Increase in respiration rate by adrenaline is coupled with disassembling of mitochondria shown in Fig. 4 for the same experiment. An increase in respiration in smaller assemblies coincides with our other observations. This inverse dependence of the rate of respiration on the size of assemblies provides evidence that decrease of respiration by sucrose is even more pronounced than the observed inhibition. Inhibition is observed instead of stimulation, which accompanies disassembling in the KCl medium. Therefore, sucrose induces considerable difference in respiration from native conditions. Adrenaline induced differences in the rate of respiration and in the size of assemblies are in a good agreement. Morphometric differences are rather high in all cases; decrease is 40–60% (Fig. 4) compared with the respiratory differences which are divided equally between increase per 15–25% and per 40–55% (Fig. 6). The higher structural difference can be due to the additions during respiration measurement, particularly, phosphate, substrate, and the ATP formed (Temnov et al., 2000a) induce disassembling and this diminishes in vitro functional regulatory difference in the state of mitochondria in vivo. However, dissipation is not instant and not complete. After phosphorylation some assemblies still can be found although they are considerably smaller and in a less number than in the homogenate stirred in the homogenization medium. This explains both phenomena, that difference in the rates of respiration between homogenates with large and small assemblies is still preserved during incubation and that this is less than in the microscopic measurements. 3.4. Respiratory ‘jump’ is related with hyperactive respiration of small assemblies It is known that just after the addition of mitochondria in the incubation medium with added substrate, the rate of respiration jumps for several seconds and

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then declines. The nature of this phenomenon, called ‘jump’, remained obscure and this is usually not calculated and considered in experiments with mitochondria. One can see from Fig. 5 that in the homogenate, in KCl medium, with succinate added, the jump is great. Simultaneous morphometric and polarographic studies show that the jump is greater during the first 1–2 h of homogenate storage. After this, the jump diminishes and remains stable for several hours more. This correlates well with selfassembling of mitochondria in the homogenate. As will be shown further in Table 2 the jump is not great with endogenous substrates. Therefore, we think that the jump of respiration is related with very active respiration of small assemblies and single mitochondria in the KCl medium. Changes in the jump during homogenate storage have no pronounced effect on the rates of respiration in States 3 and 4. Long ‘tail’ of jump can only overestimate the rate of respiration with substrate before ADP addition. The process of disassembling–assembling can also contribute to jump in mitochondria isolated in sucrose but considerably less than in KCl homogenate. We found earlier that some assemblies are also formed in concentrated suspensions of mitochondria (80–90 mg protein/ml) even in sucrose containing 10 mM Tris (Kondrashova et al., 1987). However, the structural interaction of mitochondria in pure suspension is considerably less than in the homogenate. As shown above, respiration in sucrose medium is also lowered compared with KCl. Therefore, the magnitude of jump and its changes in pure mitochondria are less than in the homogenate and are neglected. Instead, in the homogenate both phases of the jump, rapid and slow, can be measured and used as an additional test of respiration. It has an advantage over States 3 and 4 because it is measured when structural organization of the mitochondria is closer to that in the tissue than in the states, which develop later in the course of incubation when most assemblies are dissipated. 3.5. SP is better pronounced in assembled mitochondria compared with separate granules SP, i.e. guanosine triphosphate (GTP) formation coupled to KGL oxidation is undeservedly not investigated enough compared with oxidative phosphorylation. However, Olson discovered important regulatory

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role of GTP as general inhibitor of oxidation of nicotinamide adenine dinucleotide (NAD)-dependent substrates. Besides, he claimed that GTP immediately generated by SP could be of great self-importance in the regulation of mitochondrial biosynthetic processes. In these respects, the GTP cannot be replaced by ATP (Olson and Allgyer, 1971, 1973a,b). The absence of wide investigations of SP is probably due to the lack of convenient method for its measurement similar to polarographic study of oxidative phosphorylation. Olson proposed similar polarographic study of SP using the registration of inhibition of KGL oxidation by GTP formed in the course of this process. However, inhibition is hardly replicated in standard mitochondria. Oligomycin addition is necessary to ensure inhibitory effect of GTP. Obligatory addition of oligomycin for the development of respiration inhibition by GTP suggested the existence of an ATP-ase leak in mitochondrial membrane of standard mitochondria. A striking advantage of assembled MCH in homogenate is that inhibition of KGL oxidation is well observed with no oligomycin. This allows wide investigation of the physiological role of SP. Fig. 7 presents polarographic recordings of SP by inhibition of moderately uncoupled KGL oxidation by the GTP formed. Trace 1 shows that after acceleration of KGL oxidation by CCCP, spontaneous inhibition of respiration occurs. Olson reported that this inhibition blocks oxidation of all NAD-dependent substrates but not succinate. The latter is also demonstrated in this figure: inhibition is abolished by succinate addition. Thus, inhibition of KGL oxidation in assembled mitochondria is well pronounced without oligomycin. Trace 2 presents the course of KGL oxidation without inhibition. This is typical of standard mitochondria without oligomycin. However, this example also presents an experiment in the assembled mitochondria in the homogenate but taken from adrenaline administered animal. Thus adrenaline abolishes GTP inhibition. This effect coincides with reciprocal action of the reciprocal hormones, adrenaline and acetylcholine in mitochondria. Acetylcholine administration stimulates KGL oxidation and supports GTP inhibition (Shostakovskaya et al., 1986; Kondrashova and Doliba, 1989), while adrenaline stimulates succinate oxidation (Kondrashova, 1991) and inhibits KGL oxidation coupled with GTP formation. GTP inhibi-

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Fig. 7. SP in mitochondria in rat liver homogenate registered by self-inhibition of moderately uncoupled KGL oxidation in KCl prepared and incubated homogenate. KCl cold prepared and cold stored homogenate (as in Fig. 1). Traces from the left to right – control rat, adrenaline administrated rat, adrenaline with oligomycin, and control with oligomycin. Basal incubation medium and other conditions as in Fig. 6. Additions: 1.5 mM KH2PO4, 4 mM KGL, 5 £ 10 27 M CCCP, 4 mM succinate, and 1 mg/mg oligomycin.

tion can be restored in preparation from adrenalineadministered animal by oligomycin addition, as shown in trace 3, which is similar to trace 1. However, oligomycin addition eliminates difference between adrenaline and control (trace 4) animals, which is well pronounced without oligomycin. These data show that the assembled mitochondria have an important advantage over standard mitochondria: SP can be studied without oligomycin and this allows investigation of its physiological regulation. GTP inhibition of respiration as a very sensitive test of the state of mitochondrial membrane reveals alteration of mitochondria by sucrose even more clearly than succinate oxidation, shown in Fig. 5. The observation of GTP inhibition without oligomycin is possible only under preparation and incubation of homogenate in the KCl medium (as in Fig. 7). Addition of sucrose under preparation or incubation abolishes GTP inhibition. Comparison experiments were carried out in two halves of the same liver. Homogenates of one half of the same liver were prepared in KCl and of the other in the sucrose medium. KCl homogenate was incubated in KCl medium. An example of this experiment is presented in Fig. 7. Sucrose homogenate was incubated in sucrose–KCl medium. An example of this experiment with half of the same liver is presented in Fig. 8. It is clearly seen that in the presence of sucrose there is no indication of GTP inhibition without oligomycin. The rate of respiration is decreased particularly with adrenaline compared with KCl medium. This is similar to results of succinate oxidation given in Fig. 5. The

difference between control and adrenaline administered animals is not revealed in the medium with sucrose without oligomycin. Addition of oligomycin in sucrose medium leads to GTP inhibition and to incomplete restoration of high rate of respiration in adrenaline preparation. The extent of inhibition in the presence of oligomycin in sucrose is less than in KCl. Oligomycin addition probably increases at any extent the energization of mitochondria impaired by sucrose. In its presence, the acceleration of respiration in adrenaline administered animals is revealed. However, this is considerably less pronounced than in the KCl medium. Both the high rate of adrenaline induced respiration and GTP inhibition are impaired in sucrose medium compared with KCl. 3.6. Membrane potential in assembled mitochondria in homogenate The ability to maintain high membrane potential is a sensitive indication that mitochondrial membrane is intact and leaks are absent. The measurements presented in Fig. 9 show that with succinate the membrane potential reaches theoretical value which is stable during several hours of storage. 3.7. Peroxidative oxidation of lipids in assembled mitochondria The higher stability of mitochondrial membrane in assemblies can be due to better antioxidant protection in the homogenate. An estimation of products of peroxidative oxidation of lipids in the standard mito-

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Fig. 8. SP in mitochondria in rat liver homogenate registered by self-inhibition of moderately uncoupled KGL oxidation in sucrose prepared and incubated homogenate. Pair experiment in the second half of the same liver which is presented in Fig. 7. Homogenate was prepared in 250 mM sucrose, 10 mM HEPES, pH 7.5, preparation and storage in cold. Incubation in sucrose–KCl medium, 150 mM sucrose, 50 mM KCl, other additions as in Fig. 7. I, No oligomycin; II, with oligomycin; 1, control animal; 2, adrenaline administered animal. Comparison results in sucrose with those in KCl (Fig. 7) show that sucrose decreases the rate of respiration and abolishes both GTP inhibition of respiration and adrenaline stimulation of respiration.

chondria and in the homogenate during storage revealed great difference. It was determined for comparison that the mitochondrial protein consists of about 25% of total protein in the homogenate. A comparison of the level of lipid peroxidation products

in the mitochondria and homogenate during storage is given in Table 1. The data show that lipid peroxidation is one order less in the homogenate than in the mitochondria. The increase of malonic dialdehyde (MDA) in the homogenate after storage is even less

Fig. 9. Membrane potential of mitochondria in KCl rat liver homogenate during prolonged storage. Homogenate was prepared and stored as in Fig. 1. Basal incubation medium and conditions are as in Fig. 7. Succinate, 4 mM and CCCP 2.5 £ 10 27 M were used. The data demonstrate absolute stability of membrane potential during 4 h storage. This was observed also several hours longer. Membrane potential in mitochondria in homogenate is more stable than in isolated mitochondria.

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Table 1 Products of lipid peroxidation in rat liver homogenate and isolated mitochondria a Tissue

Mitochondria Freshly prepared In 3 h

MDA (nmol/mg) Per total protein

D

0.048 ^ 0.008 0.141 ^ 0.002

0.093

Homogenate (containing 1 mg of total protein and 0.25 mg of mitochondrial protein) Freshly prepared In 3 sp ¼ 0.25 . h a

MDA probably introduced with mitochondria 0.012 0.009 0.035

0.095 ^ 0.001 0.104 ^ 0.001

Determinations were made in three to four preparations from different animals.

than which could be introduced with portion of pure mitochondria. Besides the main portion of the initial MDA in the homogenate, which is two-fold greater than in pure mitochondria, is only negligibly increased after storage compared with pure mitochondria. This provides evidence that there is a considerably higher antioxidant defence in the homogenate than in pure mitochondria. These data explain why mitochondrial membranes in homogenate are more stable. 3.8. Calcium capacity of assembled mitochondria compared with standard According to our previous investigations, Ca 21 capacity is considerably a more sensitive indicator in vitro for changes in the functional state of mitochondria in vivo than ADP phosphorylation. This can be explained by several reasons. The energy load on the respiratory chain is higher for Ca 21 transport than for ADP phosphorylation. The load is increased when several additions of Ca 21 are used for Ca 21 capacity determination. Ca 21 mediates hormone regulation of mitochondria and presents these effects in vitro. Participation of Ca 21 is excluded under phosphorylation measurements. Due to this hormone regulation, which determines physiological changes in organism, cannot be observed completely. We found that Ca 21 transport measured in the mitochondria in homogenate is completely abolished by ruthenium red and malonate (data not given). Besides, values of Ca 21 capacity as determined with Ca 21 and H 1 electrodes agree with measurements by membrane potential. This provides

evidence that in rat liver homogenate Ca 21 is accumulated by mitochondria. We have elaborated three tests for Ca 21 capacity measurement: in the presence of only succinate as is conventionally used, in the presence of succinate and ADP added and phosphorylated before Ca 21. ADP addition increases Ca 21 capacity in intact animals probably at the expense of the ATP formed and prevention of membrane pore opening (Novgorodov et al., 1992; Andreeva and Crompton, 1994). The third measurement is carried out in the presence of succinate, ADP, and glutamate, which eliminates oxalacetate inhibition of succinate oxidation. The set of three tests reveals more completely the changes in the state of mitochondria. Ca 21 capacity in cold and warm KCl homogenates (prepared from two halves of the same liver) during storage is shown in Fig. 10. In this case, succinate 1 ADP were in the medium. The data provide evidence that the preparation has high Ca 21 capacity and kept this stable for at least 2 h as in this example. Ca 21 capacity is higher in the cold homogenate. A slightly lower Ca 21 capacity in the warm homogenate is probably not due to impairment of mitochondria but because in large assemblies mitochondria are less available for Ca 21. As shown above mitochondria in large assemblies have also lower rate of respiration. Nevertheless, Ca 21 accumulation proceeds well also in warm mitochondria and is stable in time. The decrease of the rates of mitochondrial functions in the sucrose medium is also observed by Ca 21 capacity. As shown in Fig. 11 sucrose addition to KCl medium progressively decreases the rate of Ca 21

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261

Fig. 10. Ca 21 capacity of mitochondria in cold and warm KCl rat liver homogenate during storage. Homogenate was prepared from two halves of the same liver under cold and warm conditions and stored under preparation temperature as in Figs. 4 and 6. A, cold preparation and storage; B, warm preparation and storage. From upper curves to bottom – freshly prepared homogenate, in 1 h, 3 h. Incubation medium – 125 mM KCl, 1 mM HEPES, pH 7.2. Succinate 4 mM, ADP 200 mM added before Ca 21, CaCl2 was added by portions per 100 nmol. Registration by Ca 21sensitive electrode.

uptake from the medium. This is evidenced by an increase of trace amplitude after Ca 21 addition. It is remarkable that relative Ca 21 capacity is greater in assembled mitochondria in the homogenate compared with standard mitochondria. An example of comparison is presented in Fig. 12. Ca 21 capacity was measured in concentrated homogenate and mitochondria obtained from another half of the same liver by

the standard method with sucrose. It was determined that the mitochondrial protein consists of 25% of the homogenate protein. In the first pair, nearly equal amounts of mitochondrial protein were taken in the homogenate and in the mitochondria. The capacity/ mg of mitochondrial protein was considerably greater in the homogenate. In the second pair, the same amount of mitochondria and a half of the homogenate

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Fig. 11. The effect of sucrose on the rate of Ca 21 accumulation. Warm stored and prepared KCl homogenate. Incubation medium as in Fig. 10, ADP was not added, KCl and sucrose were changed: 1, KCl 125 mM; 2, KCl 62.5 mM 1 sucrose 125 mM; 3, sucrose 250 mM. Registration by Ca 21 electrode. Sucrose markedly decreases the rate of Ca 21 accumulation.

amount was taken. Additionally, ADP was added before succinate. ADP increased the capacity. Under these conditions, capacity/mg of mitochondrial protein contained in sample was also higher for the homogenate. The initial deflection of records shows ADP phosphorylation. Its rate is also higher for mitochondria in the homogenate. The increase of measured processes in the mitochondria in the homogenate in percents to pure mitochondria shows that these are greater for Ca 21 capacity per 170–225% and for phosphorylation rate per 20%. The sensitivity of Ca 21 capacity test to the in vivo or in vitro induced changes in intensity of energy processes can be increased by varying the substrate concentrations. The decrease of succinate concentration or ADP omission diminishes the Ca 21 capacity and makes this more sensitive to changes in the state of mitochondria. In contrast, using complete mixture with glutamate increases the Ca 21 capacity in the mitochondria with oxaloacetate inhibition. Depending on the goal of the study, variations in energy supply for Ca 21 accumulation allow to reveal changes in the state of mitochondria escaped from phosphorylating respiration measurements. Greater Ca 21 capacity of mitochondria in the

homogenate compared to the pure mitochondria can be due to a higher stability of mitochondrial membranes as shown above and evidenced by better GTP inhibition and lower lipid peroxidation.

4. Discussion We developed a protocol for the preparation of concentrated rat liver homogenate preserving the native assemblies of mitochondria in isotonic KCl and showed that all key energy functions of the mitochondria can be investigated in such a homogenate. The energy functions in assemblies are well pronounced and are even better than in standard mitochondria dispersed into single granules in sucrose. All the measured parameters remain stable for at least 5–7 h and can be investigated even on the next day. It is known that during isolation, the mitochondria are mostly impaired at the step of homogenate. However, this is true only for diluted sucrose homogenate prepared according to the standard procedure. The properties of mitochondria in diluted homogenate are close to isolated mitochondria (Jensen et al., 1983). Concentrated, isotonic KCl homogenate is quite a different system that contains cytosol proteins, physiological concentration of potassium ions, and

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Fig. 12. Ca 21 capacity and rate of ADP phosphorylation of mitochondria in KCl homogenate and in ones isolated in sucrose. Cold prepared KCl homogenate was used and mitochondria obtained from the other half of the same liver by standard method with sucrose. It was stated previously that Ca 21 accumulation in the homogenate is completely abolished by ruthenium red or malonate and that the mitochodrial protein is 25% from the total protein of homogenate. Registration was with pH electrode. Incubation medium as in Fig. 10, but succinate 1.5 mM (this provides submaximal Ca 21 accumulation). 1,2, without ADP addition; 3,4, with ADP addition before Ca 21. Bottom row of numbers present Ca 21 capacity in nmol/mg mitochondrial protein. Numbers on the curves of ADP phosphorylation give the rate of phosphorylation in ngH 1/min mg mitochondrial protein. 1, homogenate 3.5 mg; 2, mitochondria 3.7 mg; 3, homogenate 1.75 mg; 4, mitochondria 3.7 mg. As shown equal amounts of mitochondria in the homogenate are more active than pure mitochondria.

does not contain damaging sucrose. All these preserve contact between the mitochondria and with mitochondria and reticulum. This retains some metabolic processes, particularly the ability to oxidize lactate, which is lost in dissipated mitochondria, as well as the functional differences (Gosalvez, 1996). An important advantage of homogenate used is that it contains the whole population of mitochondria in the tissue. The absence of any contact of mitochondria with sucrose during our procedure is also very important. We have observed that the rates of phosphorylating respiration and Ca 21 transport are considerably decreased by sucrose. These results agree with other data (Devin et al., 1997a; Chavez, 1997). The higher rate of mitochondrial processes in KCl is due to a larger matrix volume, which corresponds to that in cells. Sucrose diminishes the matrix volume. This effect

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can be considered as an artifact. Sucrose also changes considerably the affinity of mitochondria to basic proteins (Hillar and Rzeszski, 1965). Sucrose effect is not immediately and completely reversed after changing the medium (Devin et al., 1997a). Therefore, responses of mitochondria, which have been in contact with sucrose, are altered compared with pure KCl organelles. In our procedure, the mitochondria have no contacts with sucrose and thus preserve matrix volume closer to native than after isolation in sucrose. Simultaneous measurements of the size of assemblies and respiratory rate (Figs. 4 and 6) showed that reversible assembling serves as the factor of respiration rate regulation. The rate of respiration is low in large assemblies and high in small assemblies. As shown, KCl and warmth induce assembling while sucrose and cold induce disassembling. The inhibitory effect of sucrose on respiration is even greater than the difference in respiratory rates in KCl and sucrose medium because inhibition is observed instead of activation, which accompanies disassembling in the KCl medium. Our data revealed a new aspect of adrenaline stimulation of respiration. This includes disassembling mitochondrial network in the cell. The magnitude of adrenaline stimulation of respiration is greater in initially larger assemblies. We have also found that the enlargement (self-organization) of assemblies can be continued during storage, more intensively, under warm storage. Morphometric properties of mitochondria are in good agreement with respiration and are even more a sensitive test than phosphorylating respiration. The higher sensitivity of morphometric test is apparently due to the dissipation of mitochondrial assemblies during incubation, which we have shown in separate experiments. Therefore, mitochondria preserve their native state more completely under conditions of morphometric investigations. However, they still keep ‘print’ of their in vivo structural state also in the course of biochemical measurements, particularly in the initial minutes. The correlation between morphometric parameters found in our investigations, allows us to judge the energy functions by the size of assemblies and the rates of assembling: mitochondria in large assemblies possess higher energy control of respiration, than in the small. This structural test is sensitive and convenient, particularly for rapid clinical investigations.

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Table 2 Comparison of properties of mitochondria in KCl homogenate and in sucrose suspension after isolation in sucrose a Morphology MCH H

MCH S

90% in assemblies, mean area 10–100 mm 2, depending on the state of animal, smaller after isolation and growing during storage. Single mitochondria about 10–20% of total are present after isolation and disappear during storage joining to assemblies 100% as single granules under low concentration of protein (about 40 mg/ml) Form assemblies in concentrated suspensions (80–90 mg protein/ml) which are smaller and less stable than in homogenate

Respiration (ng-at O/min/mg protein). (Data on this and the following measurements are taken from pair experiment in which repeated measurements were identical or closely agree. Therefore, negligible deviations are not given) V2 Succinate, 4 mM MCH H 68 MCH S 16

V3

V4

RC

t (s)

2,4-DNP

192 96

76 26

2.48 3.0

11 32

172 121

68 4

96 38

32 8

3.0 5.1

15 33

124 48

Glutamate, 8 mM MCH H 32 MCH S 1

116 41

40 10

2.75 3.3

12 39

144 66

72 23

44 9

1.6 2.6

19 68

76 27

108 46

40 14

2.6 3.2

17 45

124 70

KGL, 8 mM MCH H MCH S

Malate 1 pyruvate, 5 1 5 mM MCH H 64 MCH S 2 Palmitoyl-carnitine, 50 mM MCH H 84 MCH S 3 Respiratory jump

MCH H

1–2 h 4h

MCH S

1–2 h 4h

DO

V1

SUC End substrate SUC End substrate

180 64 40 48

SUC End substrate SUC End substrate

15 12 10 9

Ca 21 capacity (nmol/mg protein) Succinate

1.5 mM

2.5 mM

4 mM

MCH H MCH S

280

450 240

540 276

22 16 10 5 18 1 15 0.3

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Table 2 (continued) Membrane potential (mV) Succinate

Suc 1 CCCP

MCH H 210 132 MCH S 185 132 SP, registered by GTP inhibition of uncoupled KGL oxidation without oligomycin Initial rate

MCH H MCH S

120 48

Final rate

48 48

% Of inhibiton ¼ GTP control 60 0

a The area of assemblies as a new structural test of energy state of mitochondria. Correlation of morphometric and bioenergetics study of mitochondria in homogenate presented here and elsewhere showed that larger assemblies correspond to the higher energy state of MCH which is typical of quiescent healthy animals and patients. Dissociation of assemblies is observed under adrenaline induced excitation or pathogenous influences. It is of importance that morphometric test is more sensitive than bioenergetics ones because conditions for microscopy are closer to the state in tissue than under biochemical measurements. Besides, morphometric measurement is very rapid and demands only a small piece of tissue, 50–100 mg.

The advantages of investigating mitochondria under preservation of their intrinsic property of selfassembling are summarized in Table 2. Data presented in the table are obtained in the homogenate and mitochondria prepared from two halves of the same liver. The data are given per mg protein of mitochondria considering that the mitochondrial protein is 25% of total protein in the homogenate. The first panel of the table shows that mitochondria in the homogenate retain blocks of mitochondrial network and the ability to assemble. This property is completely lost in widely used diluted mitochondrial suspensions and is weak in concentrated suspensions. Assemblies of mitochondria in homogenate can be used as an adequate model to study mechanisms of cytoskeleton reversible assembling and its role in metabolic control. The second panel on phosphorylating respiration presents results of a pair experiment in two halves of the same liver in homogenate and mitochondria oxidizing different substrates. As shown, succinate and different NAD-dependent substrates, including palmitate are well oxidized in the homogenate. For all substrates, the rates of States 3 and 4 respiration are considerably higher in mitochondria in homogenate, two-fold and three- to four-fold, respectively, and even greater for respiration with substrate before ADP addition. Respiratory control is higher in mito-

chondria in sucrose while ADP phosphorylation is two- to three-fold more rapid in mitochondria in the homogenate. The rate of uncoupled respiration is also higher in the homogenate. We think that the decrease of respiratory control in this case does not evidence impairment of oxidation and phosphorylation coupling because phosphorylation is evenly accelerated. The decrease of respiration is due to sucrose inhibition, while the rise in homogenate besides stimulation by KCl can also be attributed to a greater participation of ATP utilizing processes. This is closer to the state in the cell as was shown by the comparison of responses of washed mitochondria with those keeping contacts with the cytosol (Ishichara et al., 1965; Gosalvez et al., 1996). The data on respiratory jump present difference of jump in mitochondria in homogenate and in those isolated in sucrose. DO during rapid phase of jump is a convenient measure of its value. The following rate of the second slow phase is also given as V1. Jump is great in 1–2 h stored homogenate with added succinate and is considerably less with endogenous substrates. Jump is diminished considerably in the homogenate after 4 h storage and became equal with succinate and endogenous substrates. This provides evidence that the jump in the homogenate is more related with assembling of mitochondria in homoge-

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nate during storage than with oxidation of endogenous substrates. Jump in mitochondria is considerably smaller in all cases than in the homogenate. This agrees with the suggestion that this is mainly related with the assembling of mitochondria, which is weak in sucrose preparations. The next group of data on Ca 21 capacity, membrane potential, and MDA formation (presented in Table 1) provide evidence for higher stability of mitochondrial membranes in the homogenate than in sucrose isolated mitochondria. Ca 21 capacity was compared with concentrations of SUC providing submaximal and maximal Ca 21 accumulation. In both cases, the accumulation was nearly two-fold higher in mitochondria in the homogenate. Membrane potential in the mitochondria in the homogenate is also higher and near to the maximal level. The validity of difference between pure mitochondria and the homogenate in the energized state is confirmed by the fact that the membrane potential falls to the same level in uncoupled state. As shown above, the peroxidative oxidation of lipids is considerably less in mitochondria in the homogenate than in pure mitochondria. The most important property of mitochondria in the homogenate is considerably more complete SP leading to NAD-dependent substrate inhibition by GTP. This allows the investigation of this important mechanism as widely as oxidative phosphorylation. Particularly this allows investigation of the physiological regulation of GTP control by adrenaline, which is masked by oligomycin (presented in Figs. 7 and 8). The completeness of GTP inhibition depends greatly on the fine changes in intact state of the inner mitochondrial membrane. To support this, addition of oligomycin or increase of tonicity is necessary in the mitochondria. Therefore, completeness of SP and GTP control in the homogenate can also provide evidence that the mitochondrial membranes are more intact. The data shown provide evidence that the investigation of mitochondria in the homogenate allows to avoid impairment of mitochondria by dilution, washing, and addition of sucrose and to provide stability during storage for 5–7 h and preservation energy properties for 24 h of the total population, including swollen fraction.

The investigation of mitochondria in the homogenate opens new possibility of using morphometry of assemblies as self-dependent sensitive test of the energy state of mitochondria in the organism; the larger assemblies have lower respiration and provide evidence of quiescent state of mitochondria, while smaller assemblies have higher respiration, and provide evidence of the transition to activity or pathology. Rapidity and simplicity of preparation procedure and possibility to obtain good preparations from small amounts of tissue up to 100 mg for investigation of the energy processes and even smaller for morphometry are most useful for clinical investigations, whose number is growing in connection with the study of mitochondrial diseases. Acknowledgements For many years our attempts to isolate assemblies of mitochondria, which better preserve in vitro properties of mitochondria in vivo, were supported by the stimulating interest of Britton Chance. Presentation of part of this work at the Seminar at Johnson Research Foundation was important for M.N. Kondrashova. We are grateful to B. Chance for support and for the laboratory name of assemblies of mitochondria ‘Good mitochondria’. The work is supported by the Grant of Russian Foundation for Fundamental Investigations For Leading Scientific Schools 00-15-97847. References Andreeva, L., Crompton, M., 1994. An ADP-sensitive cyclosporinA-binding protein in rat liver mitochondria. Eur. J. Biochem. 221, 211–218. Bailey, N., 1963. Statistical Methods in Biology, Mir, Moscow. Baquet, A., Hue, L., Meijer, A.J., van Woerkom, G.M., Plomp, P.J., 1990. Swelling of rat hepatocytes stimulates glycogen synthesis. J. Biol. Chem. 265, 955–959. Chance, B., Williams, G.R., 1955. Respiratory enzymes in oxidative phosphorylation. I. Kinetics of oxygen utilization. J. Biol. Chem. 217, 383–393. Chavez, E., Moreno-Sanches, R., Zazueta, C., Rodrigues, J.S., Bravo, C., Reyes-Vivas, G., 1997. On the protection by inorganic phosphate of calcium-induced membrane permeability transition. J. Bioenerg. Biomembr. 29, 571–577. Devin, A., Guerin, B., Rigoulet, M., 1996. Dependence of flux size and efficiency of oxidative phosphorylation on external osmolarity in isolated rat liver mitochondria: role of adenine nucleotide carrier. Biochim. Biophys. Acta 1273, 13–20.

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