CRYOBIOLOGY
28, 445453
The Effects
(191)
of Cryopreservation on Protein Synthesis and Membrane Transport in Isolated Rat Liver Mitochondria
PETER DE LOECKER, BARRY J. FULLER,* AND WILLIAM DE LOECKER Afdeling Biochemie, Faculteit Geneeskunde, Katholieke Universiteit te Leuven, B-3000 Leuven, Belgium, and *Academic Department of Surgery, Royal Free Hospital School of Medicine, University of London, London NW3 2QG, England Protein synthesizing activity and membrane transport were examined in fresh and cryopreserved isolated rat liver mitochondria. In the presence of 0.6, 1.2, and 1.8 M final concentrations of dimethyl sulfoxide (Me,SO), both metabolic parameters were considerably inhibited in the fresh samples and even more inhibited in the cryopreserved specimens. However, simple exposure to this penetrating cryoprotectant, followed by its subsequent removal by washing, did not seem to affect significantly the examined functions. When different freeze-thaw regimes were investigated, it was observed that optimal recovery of protein synthesis and membrane transport functions were obtained when fast freezing took place in the absence of Me,SO. B ~1 Academic PXSS, IIIC.
With the aim of the eventual successful cryopreservation of liver cells or tissue, several studies have been undertaken to examine hepatic mitochondrial structure and behavior after freezing and thawing (1, 5, 9-11, 14, 23, 24, 28, 29). Damage occurring during cryopreservation has been accompanied by partial uncoupling of oxidative phosphorylation (9, 14). In recent studies, mitochondria cryopreserved in a dimethyl sulfoxide (Me,SO)containing medium, although presenting a general decrease in efficiency of substratelinked oxygen uptake, did not show gross morphological damage or dramatic uncoupling. Thus slow cooling and the addition of the penetrating cryoprotectant Me,SO to some extent protected structural integrity as well as the electron transport chain. As ATP synthesis in these circumstances remained intact, the ATPase system appeared unaffected by the freeze-thaw cycle (14). Apart from these metabolic characteristics, mitochondria also present a specific
protein-synthesizing activity distinct from the process which occurs in the cytosolic ribosomal compartment. Apart from the nuclear-encoded proteins synthesized on extramitochondrial polyribosomes and transferred or imported into the mitochondria as a necessity for their adequate functioning, mitochondrial-encoded proteins synthesized on intramitochondrial ribosomes are equally required to serve as structural compounds (17, 21). Electrophoresis of submitochondrial particles of most species revealed only a limited number of polypeptide bands consistent with the low informational content of mitochondrial DNA (21, 22). Nevertheless, these specific proteins are important for normal organelle processes. Thus intramitochondrial protein synthesizing activity, important to long-term function and structure, was analyzed in order to evaluate the possible cryoinjury occurring to this system during the freeze-thaw cycle. MATERIALS
Mitochondrial
Received March 14, 1990; accepted December 28, 1990
Mitochondria
AND METHODS
Preparation
were prepared from livers
445
0011-2240191 $3.00 Copyright All rights
8 1991 by Academic Press, Inc. of reproduction in any form reserved.
446
DE
LOECKER,
FULLER,
of male Wistar R rats, weighing 200-250 g. The animals were fasted overnight. The livers were removed and washed in KrebsRinger bicarbonate buffer. All further preparative procedures were performed at 4°C. Liver tissue (5 g) was homogenized by hand in 50 ml of homogenization medium, pH 7.8, containing 250 mM sucrose (Merck, Darmstadt, Germany), 10 mM TrisChloride (Merck), 1.0 mM EDTA (Calbiothem, Los Angeles) using a glass Potter Elvehjem homogenizer with a Teflon pestle. After centrifugation for 10 min at 600g to remove debris the pellet was resuspended in an equal volume of buffer and spun again. This procedure was repeated and both pooled supernatants were centrifuged at 10,000g (Sorvall, RCZB, Sorvall Inc., Norwalk, CT) for 10 min. The ensuing mitochondrial pellet was gently resuspended by hand with a loose-fitting glass homogenizer in an equal volume of medium and recentrifuged at 10,OOOg for 10 min. This procedure was repeated once more (2). This final precipitate of washed mitochondria was resuspended in 25 ml of homogenization buffer, thus obtaining a final concentration of about 24 mg of mitochondrial protein per milliliter of medium. Protein concentrations were determined by the folin phenol method (20). Cryopreservation The mitochondrial suspension was exposed for 30 min at 4°C to a final concentration of 1.8 A4 of dimethyl sulfoxide (Me,SO, Merck) which was added slowly under gentle agitation as described (14). For the analysis of fresh mitochondria and some frozen groups, the Me,SO-containing mitochondrial suspension was subsequently diluted 10 times with homogenization buffer, and after centrifugation at 10,OOOg for 10 min, the pellet was resuspended in the original volume of fresh medium. The samples to be cryopreserved with or without Me,SO were placed in poly-
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propylene tubes (Nunc Cryotubes 7 x 1.2 cm, Denmark) and progressively slowly cooled at l”C/min in an alcohol bath (Fryka-Therm-FT 800, Copenhagen, Denmark) to -7°C when nucleation was induced by clamping the tubes with cold forceps. After further cooling at l”C/min to -36°C the lowest limit attainable by the Fryka-Therm, the samples were placed in liquid N, gas phase for 30 min and subsequently submerged, without cooling rates being estimated. In other experiments the mitochondrial suspensions with or without Me,SO were fast cooled by immediate submersion in liquid nitrogen. After cryopreservation for 1-3 days, thawing took place in a waterbath at 37°C. The cryoprotectant was removed as for the nonfrozen samples by dilution and centrifugation followed by resuspension in the original volume of fresh medium (14). Incubation Procedure From these final suspensions of fresh and cryopreserved mitochondria 0.2 ml was added to 0.8 ml of incubation buffer at pH 7.6 consisting of 90 mM KC1 (Merck), 50 mM N-(Tris-(hydroxymethyl)methyl)glycine (Tricine) (Merck), 10 m&f MgCl, (Merck), 5 m&f potassium phosphate buffer (Merck), 1 mM EDTA (Calbiochem), and as energy source: 2 m&f ATP (Sigma, St. Louis, MO.), 5 m&f phosphoenolpyruvate (Sigma), and 10 pg/ml pyruvate kinase (Sigma). Each 100 ml of incubation buffer equally contained 1 ml of an L-amino acid supplement (Vamin, Vitrum, Stockholm 12, Sweden). To each final 1 ml of incubation medium containing the mitochondria 1 pCi of L-[U-‘4C]isoleucine (sp radioact: 300 mCi/mmol; Amersham International, Amersham, Bucks, UK) was added to follow the incorporation into proteins during incubation in a shaking waterbath at 25°C over a period of up to 60 min. To follow the amino acid uptake into the mitochondria as a measure of membrane transport activity, incu-
MITOCHONDRIAL
FUNCTION
bation with 1 pCi of L-[U-14Clisoleucine was performed in the presence of 200 pg/ml of chloramphenicol (Sigma), completely blocking protein synthesis (3, 22). In some experiments, the final mitochondrial suspensions were incubated in the presence of 0.6, 1.2, or 1.g M of supplementary addition of Me,SO. All media used were passed through a 0.22~p.m Nalgene disposable filter (Nalge Cy., Rochester, NY) before incubation was started to avoid the possibility of bacterial contamination. Radioisotope Evaluation After incubation of fresh and cryopreserved mitochondria with L-[U-14C]isoleucine, protein synthesis was arrested by adding 5 ml of trichloroacetic acid (TCA, Merck) 20%. After three more washes with 10 ml of TCA, the precipitated proteins were dissolved in 2 ml of Lumasolve (Lumac Systems AG, Basel, Switzerland) by heating overnight at 60°C. After the addition of 10 ml of Lipoluma (Lumac Systems AG), the incorporated radioactivity was counted in a liquid scintillation spectrometer (Rack Beta, LKB, Wallac, OY , Turku, Finland). For membrane transport experiments amino acid uptake was stopped after incubation by the addition of 10 ml of fresh icecold incubation medium containing 30 mmol of nonradioactive isoleucine (Sigma). After centrifugation at 10,OOOg for 10 min, followed by three identical washes with fresh isoleucine-containing medium, the precipitated mitochondria were dissolved in Lumasolve and were further processed to evaluate the accumulation of radiolabeled amino acid. Radioactive measurements (after subtraction of the zero time values serving as control blanks) were expressed as disintegrations per minute (DPM) per milligram of mitochondrial proteins 2 SEM. Statistical evaluation of significance was carried out according to Student’s t test.
AFTER
447
CRYOPRESERVATION
RESULTS
Effect of Me,SO on Protein Synthesis and Amino Acid Transport of Fresh Mitochondria The results of the experiments to study protein synthesis in response to Me,SO exposure are shown in Fig. 1. The incorporation of L-[U-‘4C]isoleucine into the proteins of fresh mitochondria, not previously exposed to Me,SO but equally treated by centrifugation and dilution as for the exposed
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FIG. 1. Effect of Me,SO on the incorporation of L[U-‘4C]isoleucine into the proteins of fresh mitochondria. Mitochondria were prepared as described under Materials and Methods. Mitochondriaf suspension (0.2 ml) was added to 0.8 ml of incubation medium and incubated at 25°C for up to 60 min in the presence of 1pCi L-[U-r4C]isoleucine (specific radioact: 300 mCi/ mmol) and of an energy source. Apart from the sample containing fresh mitochondria (0) Me,SO was added to the incubation samples in final concentrations of 0.6 (0), 1.2 (m), and 1.8 M (0). The amino acid incorporation into the mitochondrial proteins is expressed as disintegrations per min (DPM). Each value represents the average of six experiments k SEM.
448
DE LOECKER,
FULLER.
samples, progressed linearly over the incubation period of 60 min at 25°C. After exposure to 1.8 M Me,SO for 30 min, the washed mitochondria were pelleted, resuspended in an original volume of homogenization medium, and aliquoted 0.2 ml in 0.8 ml of incubation medium, resulting in a residual Me,SO concentration of less than 0.05 M. These exposed mitochondria did not show significantly different incorporation pattern from the fresh sample upon incubation. As increasing concentrations of Me,SO were added to the incubation buffer in which the mitochondria were suspended, incorporation of the amino acid progressively decreased. Linearity was still observed in the presence of 0.6 M Me,SO but after 60 min of incubation incorporation amounted to 75% (p < 0.001 compared to controls). In the presence of 1.2 and 1.8 M Me,SO the linear phase of incorporation lasted only 30 and 20 min, respectively, while after 60 min of incubation, compared to the samples not containing any additional Me,SO, the incorporated isoleucine amounted to 50% (P < 0.001) and 28% (P < O.OOl), respectively, with the differences becoming more marked as incubation progressed. In Fig. 2 similar studies on Me$O exposure as it affects membrane amino acid transport is shown. In the presence of chloramphenicol, blocking protein synthesis, membrane transport was evaluated by measuring the r,-[U-14C]isoleucine uptake by the mitochondria. Amino acid uptake by fresh mitochondria progressed linearly up to 10 min followed by a leveling off with only a slow increase up to 40 min. Without any addition of Me,SO to the incubation medium, but only with the residual Me,SO present after exposure to the cryoprotectant, amino acid uptake presented an identical pattern as observed with fresh mitochondria. After the addition of 1.8 M Me,SO to the incubation medium containing the mitochondria, a linear uptake con-
AND DE LOECKER 2500 r
lncubahon
time
m mm
FIG. 2. Effect of Me,SO on the L-[U-‘4C]isoleucine uptake by fresh mitochondria. Mitochondria prepared as in Fig. 1 were incubated in the presence of 200 &ml chloramphenicol. After incubation for up to 40 min at 25”C, the control samples (0) and the samples containing a final concentration of 1.8 M Me,SO (0) received 10 ml of fresh incubation buffer containing 30 mmol of nonradioactive isoleucine by which label up take was arrested. After repeated washes the samples were prepared for radioactive counting. Each value represents the average uptake values of six experiments in DPM * SEM.
tinued for 10 min although only reaching 89% of the control samples and equally leveling off to a value of 80% after 40 min of incubation (P < 0.001). The uptake values in the presence of intermediate 0.6 and 1.2 M Me,SO concentrations all fell in between the two illustrated curves (data not shown). Effect of Cryopreservation on L-[ U-14C]isoleucine Incorporation and Uptake The various freezing regimes did not significantly affect the incorporation rates of L-[U-14C]isoleucine into the proteins of cryopreserved mitochondria during the initial 10 min of incubation. However as incu-
MITOCHONDRIAL
FUNCTION
bation progressed, fast cooling achieved by immediate submersion of the mitochondria into liquid nitrogen without the cryoprotection of Me,SO resulted in the recovery of 60% of incorporation activity. In contrast the presence of Me,SO during cooling only resulted in 30% of isoleucine incorporation compared to the nonfrozen samples evaluated after longer incubation (60 min) (P < 0.001). Using the slow cooling method in the presence of 1.8 M of Me,SO the isoleucine incorporation amounted to 40% of the fresh control mitochondria after 60 min of incubation while slow cooling without Me,SO resulted in 52% of amino acid incorporation. Without the cryoprotectant Me,SO, fast cooling already resulted in a significantly higher incorporation of isoleutine after 20 min of incubation, while in the presence of Me,SO slow cooling proved beneficiary when incubation was prolonged beyond 30 min (Fig. 3). The values in mitochondria were always higher in the absence of Me,SO (fast, P < 0.001; slow, P < 0.001) than when the cryoprotectant was present during cryopreservation. When membrane transport activity was examined after thawing, the cryopreserved mitochondria presented a linear isoleucine uptake in less than 10 min, achieving a maximal uptake which remained almost constant for up to 30 min of incubation (Fig. 4). When evaluating the L-[U-14C]isoleucine uptake, there were no obvious differences between fast- and slow-cooling rates in the absence of Me,SO. In both circumstances uptake values were significantly higher than that achieved by mitochondria exposed to Me,SO during cooling (P < 0.01 in each case). With Me,SO present during slow cooling, the uptake values during subsequent incubation appeared slightly higher. In samples frozen without Me,SO the L-[U-14C]isoleucine uptake after 30 min of incubation amounted to 28% of the fresh nonfrozen mitochondria, while freezing in the presence of Me,SO generally reduced
AFTER
449
CRYOPRESERVATION
SOOOr
VI
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60
Incubation
time
in min
3. Incorporation of L-[U-i4C]isoleucine into the proteins of fresh and cryopreserved mitochondria. Preparation, incubation, and processing of the fresh mitochondria (0) was carried out as for Fig. 1. Freezing took place in the presence of 1.8 M Me,SO following a fast cooling rate achieved by immediate submersion into liquid nitrogen (A) or by a slow cooling rate, initially 1°C per min up to -36°C followed by exposure for 30 min in the N, gasphase and subsequent submersion (A). Other samples were frozen without Me,SO either quickly (m) or slowly (0). Incubation was carried out after the removal of the cryoprotectant by dilution or after analogous dilution of the samples not being exposed to Me,SO. Each value represents the mean of six experiments ? SEM. FIG.
the amino acid uptake by a further 8 to 12% (Fig. 4). DISCUSSION
The in vitro amino acid incorporation into proteins by isolated mitochondria, dependent on enzyme systems involved in oxidative phosphorylation, is generally supported by an ATP-regenerating system (18, 26, 30). However, high-energy intermedi-
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FULLER.
F% 5
10
20 Incubation
time
31 in min
FIG. 4. L-[U-“‘C]isoleucine uptake by fresh and cryopreserved mitochondria. Preparation and incubation of fresh mitochondria was carried out as for Fig. 2. Freezing and thawing was carried out as for Fig. 3. Apart from fresh nonfrozen samples (O), mitochondria were frozen in the presence of 1.8 M Me,SO according to a fast cooling (A) or a slow cooling (A) regimen. Samples to be frozen without Me,SO were subjected to either fast cooling (W) or slow cooling (0). Each value represents the mean of six experiments 2 SEM.
ates of oxidative phosphorylation generated by electron transport or ATP hydrolysis may not always be the exclusive source of energy for mitochondrial protein synthesis, as this complex mechanism appears to be governed by several energy-dependent processes (30). Analyzing the in vitro proteinsynthesizing activity, sometimes apparently conflicting observations have emerged concerning ideal conditions and circumstances in which these metabolic functions should be measured. Although ATP generated by respiration or transported across the membrane is essential for
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optimal amino acid incorporation, the necessity or advisability of adding exogenous adenine nucleotides or ATP-regenerating systems has been queried (3, 21, 22). Furthermore, depending on the source of the mammalian mitochondria from different organs, or organelles originating from microorganisms, yeasts, fungi, or plants, the advised experimental conditions may greatly vary (3, 15, 16, 19, 22). As we intend to compare metabolic activities, without trying to improve experimental conditions or to explore the complex reactions of mitochondrial protein synthesis, a well-proven method has been basically followed (2). In all instances the incubation medium contains ATP and an ATP-regenerating system. Since the amino acid incorporation curves present an initial linear ATPdependent progression, and all solutions were passed through a 0.22 pm filter before use, bacterial growth interference may be excluded (30). As no transport mechanism for a-amino isobutyric acid has been demonstrated in isolated mitochondria, amino acid transport activity was evaluated in our study by observing L-[U-‘4C]isoleucine uptake after blocking the protein-synthesizing activity by chloramphenicol (4, 8). Amino acid uptake appears to reach maximal levels during the initial 10 min of incubation for fresh organelles. Although some authors report a rapid decline of intramitochondrialfree amino acid levels on continuing incubation, this observation has not always been confirmed (3,4, 12, 30). Blocking protein synthesis and allowing membrane transport to take place affect the efficiency and the general pattern of the transport mechanism. Initial accumulation of precursor amino acids may well induce a concentration-driven self-limiting transport activity. Addition of chloramphenicol to block protein synthesis considerably reduces the absolute values of transport activity presumably because the amino acids are not being consumed in the production of new
MITOCHONDRIAL
FUNCTION
AFTER CRYOPRESERVATION
451
polypeptide chains (8). The cryopreserved leads to morphological and functional modmitochondria particularly appear affected ifications, it still appears least damaging to by chloramphenicol and present persisthe protein-synthesizing activity. As memtantly lower uptake values than to be ex- brane transport and protein synthesis bepected from the protein synthesis expericome inhibited when incubation takes place ments. in the presence of Me,SO, these mitochonIndiscriminate repeated freezing and drial functions appear best preserved when thawing seriously damages mitochondrial freezing takes place in a medium without protein synthesizing activity thus reducing Me,SO. Furthermore it appears that withthe isoleucine incorporation (21, 25). Cryoout Me,SO present, the normal advantages preservation leads to partial loss of the cy- of a slow cooling rate disappear. In our systochrome C pool released from the inner tem ATP and an ATP-regenerating system mitochondrial membrane into the incubaare both provided to fresh and cryopretion medium through the damaged outer served mitochondria, so that cryoinjury to membrane. This enzyme depletion affected the protein synthesizing activity appears by different freeze-thaw regimes is at least primarily to occur at the levels of structure one of the causes of inhibited mitochondrial or membrane organization (9, 15, 23). electron transport chain activities (23). AlApart from cryoinjury some other forms though Me,SO provides protection to the of chemical toxicity inhibit oxidative phosmitochondrial substrate-linked oxygen con- phorylation and also increase mitochonsumption, sole exposure to and dilution of drial membrane permeability (6). In rat the cryoprotectant already results in some brain mitochondria chemical inhibition of changes of mitochondrial function (7, 11, protein synthesis does not directly affect 14, 21). Reduced efficiency of oxygen up- ATP synthesis. Cryoinjury appears to aftake is already observed at concentrations fect protein synthesis on a nonspecific basis of around 0.1 M of Me,SO while inhibitory as already observed in other conditions. effects on protein synthesis only appear at Chemically induced modifications in the much higher levels. As oxidative phosrate of mitochondrial protein synthesis rephorylation is much more sensitive to sult in a nonselective stimulatory or inhibicryoinjury this process shows evidence of tory effect on the protein fractions formed serious impairment long before ultrastruc(19). The stimulatory effects of cold exposure on in viva rat liver mitochondrial proture and protein metabolism are affected (9). Since Me,SO has been observed to tein synthesis by the mechanism of instimulate mitochondrial ATP production creased triiodothyronine levels equally (possibly by equilibrium shifts between in- take place without specifically affecting volved enzyme systems) the observed in- any of the major proteins derived from mihibitory effects are unlikely to be due to tochondrial DNA (16). For the present adequate cryopreservainterference at this bioenergetic level (27). Mitochondrial protein synthesis proves far tion of functional integrity of membrane activity more resistant to freeze-thaw injury than transport and protein-synthesizing in isolated mitochondria remains problemthat seen in the original intact hepatocyte from which they are derived (13). The pro- atic and appears to be optimally achieved tection of isolated mitochondria compared during fast cooling without the addition of to the mitochondria frozen in situ during an additional permeating cryoprotectant. cooling without Me,SO may well be due to ACKNOWLEDGMENTS the relatively high sucrose levels present during preparation and cryopreservation The authors are indebted to the Belgian National Foundation for Medical Research (F.G.W.O.) for a (14). Although fast freezing without Me,SO
452
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FULLER,
grant to the laboratory, to the Nationale Loterij for a research grant, and to Mrs. F.De Wever for her valuable technical assistance.
AND
DE LOECKER
13. Fuller, B. J., Grout, B. W., and Woods, R. J. Biochemical and ultrastructural examination of cryopreserved hepatocytes in rat. Cryobiology 19, 493-502
REFERENCES
1. Araki, T. Freezing injury in mitochondrial membranes. I. Susceptible components in the oxidation systems of frozen and thawed rabbit liver mitochondria. Cryobiology 14, 144-150 (1977). 2. Beattie, D. S. Yeast versus mammalian mitochondrial protein synthesis. In “Methods in Enzymology” (S. Fleischer and L. Packer, Eds.), Vol. 56, pp 17-27. Academic Press, New York, 1979. 3. Beattie, D. S., and Ibrahim, N. G. Optimal conditions for amino acid incorporation by isolated rat liver mitochondria: Stimulation by valinomycin and other agents. Biochemistry 12, 176 180 (1973). 4. Buchanan, J., Popovitch, J. R., and Tapley, D. F. Leucine transport by rat liver mitochondria in vitro. Biochim. Biophys. Acta 173, 532-539 (1969). 5. De Giorgi, C., and Siculella, L. Stimulation of protein synthesis in isolated mammalian mitochondria by a factor in the cytosol. Cell Biol. Znt. Rep. 8, 587-590 (1984). 6. De Wit, R. H., and Brabec, M. J. Protein synthesis by hepatic mitochondria isolated from carbon tetrachloride-exposed rats. Biochim. Biophys. Acra 824, 256-261 (1985). 7. Dickinson, D., Misch, M., and Drury, R. Dimethyl sulfoxide protects tightly coupled mitochondria from freezing damage. Science 156, 1738-1739 (1967). 8. Finzi, E., Clejan, L., and Beattie, D. S. Effect of temperature on protein synthesis and leucine transport by yeast mitochondria. Me&r. Biothem. 5, 291-307 (1985). 9. Fishbein, W. N., and Griffin, J. L. Studies on the mechanism of freezing damage to mouse liver. IV. Effects of ultrarapid freezing on structure and function of isolated mitochondria. Cryobiology 13,542-556(1976). 10. Fleischer, S. Long-term storage of mitochondria to preserve energy-linked functions. In “Methods in Enzymology” (S.Fleischer and L.Packer, Eds.), Vol. 55, pp. 28-32. Academic Press, New York, 1979. 11. Foissner, I. Septation and fragmentation in Oedogonium mitochondria as different and independent effects of dimethyl sulfoxide (DMSO) treatment. Experientia 42, 958-%0 (1986). 12. Freedland, R. A., Crozier, G. L., Hicks, B. L., and Meijer, A. J. Arginine uptake by isolated rat liver mitochondria. Biochim. Biophys. Acra 802,407--112
(1984).
(1982).
14.
Fuller, B. J., Rubinacci, A., Geboes, K., and De Loecker, W. The bioenergetics of mitochondria after cryopreservation. Cryobiology 26, 333-
15.
Gadaleta, M. N., Minervini, G. R., Renis, M., De Giorgi, C., and Giovine, A. Mitochondrial DNA, RNA and protein synthesis in normal and hypothyroid developing rat liver. Cell Differ. 19, 4349 (1986). Goglia F., Liverini, G., Lanni, A., and Barletta, A. Mitochondrial DNA, RNA and protein synthesis in normal, hypothyroid and mildly hyperthyroid rat liver during cold exposure. Mol. Cell. Endocrinol. 55, 141-147 (1988). Hay, R., Bohni, P., and Gasser, S. How mitochondria import proteins. Biochim. Biophys. Acta 779, 65-87 (1984). Kroon, A. M. Protein synthesis in mitochondria. II. A comparison of mitochondria from liver and heart with special reference to the role of oxidative phosphorylation. Biochim. Biophys. Acta 91, 145-154 (1964). Kuznetsov, D. A., and Musajev, P. I. 0. Chemical-induced modulation of ATP and protein synthesis processed inside rat brain mitochondria. ht. J. Neurosci. 38, 331-343 (1988). Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein measurement with the Fohn Phenol reagent. J. Biol. Chem. 193,
340 (1989).
16.
17.
18.
19.
20.
265-275(1951).
Mills, N. C., Durwood, B. R., Littlejohn, R. A., Horst, I. A., and Kowal, J. Optimalization of in vitro protein synthesis by isolated mouse adrenal mitochondria. Anal. Biochem. 138, l&180 (1984). 22. Mockel, J. J., and Beattie, D. S. Optimal conditions for studies of amino acid incorporation in vitro by isolated skeletal muscle mitochondria. Arch. Biochem. Biophys. 167, 301-310 (1975). 23. Petrenko, A. Y., and Subbota, N. Inhibition of the mitochondrial electron transport chain by low temperatures: Losses of cytochrome C. Cryo 21.
Lett.
7, 395-2
(1986).
Privitera, C. A., Greiff, D., Strength, D. R., Anglin M., and Pinkerton, H. Oxidative phosphorylation by mitochondrial suspensions after freezing and storage at low temperatures. J. Biol. Chem. 233, 526527 (1958). 25. Roodyn, D. B. Further study of factors affecting amino acid incorporation into protein of isolated mitochondria. Biochem. J. 97, 782-793 24.
(1965).
MITOCHONDRIAL
FUNCTION
26. Roodyn, D. B., Reis, P. J., and Work, T. S. Protein synthesis in mitochondria: Requirements for the incorporation of radioactive amino acids into mitochondrial protein. Biochem. J. 80, % 21 (l%l). 27. Sakamoto, J. Effect of dimethyl sulfoxide on ATP synthesis by mitochondrial soluble F,-ATPase. J. Biochem. 96, 483-487 (1984). 28. Sherman, J. K. Correlation in ultrastructural cryoinjury of mitochondria with aspects of their
AFTER CRYOPRESERVATION
453
respiratory function. Exp. Cell. Res. 66, 371 384 (1971). 29. Tsvetkov, T., Tsonev, L., Meranzov, N., and Minkov, I. Functional changes in mitochondrial properties as a result of their membrane cryodestruction. Cryobiology 22, 47-54 (1985). 30. Wheeldon, L. W., and Lehninger, A. L. Energylinked synthesis and decay of membrane proteins in isolated rat liver mitochondria. Biochemistry 5, 3533-3545 (1966).
28, 445453
The Effects
(191)
of Cryopreservation on Protein Synthesis and Membrane Transport in Isolated Rat Liver Mitochondria
PETER DE LOECKER, BARRY J. FULLER,* AND WILLIAM DE LOECKER Afdeling Biochemie, Faculteit Geneeskunde, Katholieke Universiteit te Leuven, B-3000 Leuven, Belgium, and *Academic Department of Surgery, Royal Free Hospital School of Medicine, University of London, London NW3 2QG, England Protein synthesizing activity and membrane transport were examined in fresh and cryopreserved isolated rat liver mitochondria. In the presence of 0.6, 1.2, and 1.8 M final concentrations of dimethyl sulfoxide (Me,SO), both metabolic parameters were considerably inhibited in the fresh samples and even more inhibited in the cryopreserved specimens. However, simple exposure to this penetrating cryoprotectant, followed by its subsequent removal by washing, did not seem to affect significantly the examined functions. When different freeze-thaw regimes were investigated, it was observed that optimal recovery of protein synthesis and membrane transport functions were obtained when fast freezing took place in the absence of Me,SO. B ~1 Academic PXSS, IIIC.
With the aim of the eventual successful cryopreservation of liver cells or tissue, several studies have been undertaken to examine hepatic mitochondrial structure and behavior after freezing and thawing (1, 5, 9-11, 14, 23, 24, 28, 29). Damage occurring during cryopreservation has been accompanied by partial uncoupling of oxidative phosphorylation (9, 14). In recent studies, mitochondria cryopreserved in a dimethyl sulfoxide (Me,SO)containing medium, although presenting a general decrease in efficiency of substratelinked oxygen uptake, did not show gross morphological damage or dramatic uncoupling. Thus slow cooling and the addition of the penetrating cryoprotectant Me,SO to some extent protected structural integrity as well as the electron transport chain. As ATP synthesis in these circumstances remained intact, the ATPase system appeared unaffected by the freeze-thaw cycle (14). Apart from these metabolic characteristics, mitochondria also present a specific
protein-synthesizing activity distinct from the process which occurs in the cytosolic ribosomal compartment. Apart from the nuclear-encoded proteins synthesized on extramitochondrial polyribosomes and transferred or imported into the mitochondria as a necessity for their adequate functioning, mitochondrial-encoded proteins synthesized on intramitochondrial ribosomes are equally required to serve as structural compounds (17, 21). Electrophoresis of submitochondrial particles of most species revealed only a limited number of polypeptide bands consistent with the low informational content of mitochondrial DNA (21, 22). Nevertheless, these specific proteins are important for normal organelle processes. Thus intramitochondrial protein synthesizing activity, important to long-term function and structure, was analyzed in order to evaluate the possible cryoinjury occurring to this system during the freeze-thaw cycle. MATERIALS
Mitochondrial
Received March 14, 1990; accepted December 28, 1990
Mitochondria
AND METHODS
Preparation
were prepared from livers
445
0011-2240191 $3.00 Copyright All rights
8 1991 by Academic Press, Inc. of reproduction in any form reserved.
446
DE
LOECKER,
FULLER,
of male Wistar R rats, weighing 200-250 g. The animals were fasted overnight. The livers were removed and washed in KrebsRinger bicarbonate buffer. All further preparative procedures were performed at 4°C. Liver tissue (5 g) was homogenized by hand in 50 ml of homogenization medium, pH 7.8, containing 250 mM sucrose (Merck, Darmstadt, Germany), 10 mM TrisChloride (Merck), 1.0 mM EDTA (Calbiothem, Los Angeles) using a glass Potter Elvehjem homogenizer with a Teflon pestle. After centrifugation for 10 min at 600g to remove debris the pellet was resuspended in an equal volume of buffer and spun again. This procedure was repeated and both pooled supernatants were centrifuged at 10,000g (Sorvall, RCZB, Sorvall Inc., Norwalk, CT) for 10 min. The ensuing mitochondrial pellet was gently resuspended by hand with a loose-fitting glass homogenizer in an equal volume of medium and recentrifuged at 10,OOOg for 10 min. This procedure was repeated once more (2). This final precipitate of washed mitochondria was resuspended in 25 ml of homogenization buffer, thus obtaining a final concentration of about 24 mg of mitochondrial protein per milliliter of medium. Protein concentrations were determined by the folin phenol method (20). Cryopreservation The mitochondrial suspension was exposed for 30 min at 4°C to a final concentration of 1.8 A4 of dimethyl sulfoxide (Me,SO, Merck) which was added slowly under gentle agitation as described (14). For the analysis of fresh mitochondria and some frozen groups, the Me,SO-containing mitochondrial suspension was subsequently diluted 10 times with homogenization buffer, and after centrifugation at 10,OOOg for 10 min, the pellet was resuspended in the original volume of fresh medium. The samples to be cryopreserved with or without Me,SO were placed in poly-
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propylene tubes (Nunc Cryotubes 7 x 1.2 cm, Denmark) and progressively slowly cooled at l”C/min in an alcohol bath (Fryka-Therm-FT 800, Copenhagen, Denmark) to -7°C when nucleation was induced by clamping the tubes with cold forceps. After further cooling at l”C/min to -36°C the lowest limit attainable by the Fryka-Therm, the samples were placed in liquid N, gas phase for 30 min and subsequently submerged, without cooling rates being estimated. In other experiments the mitochondrial suspensions with or without Me,SO were fast cooled by immediate submersion in liquid nitrogen. After cryopreservation for 1-3 days, thawing took place in a waterbath at 37°C. The cryoprotectant was removed as for the nonfrozen samples by dilution and centrifugation followed by resuspension in the original volume of fresh medium (14). Incubation Procedure From these final suspensions of fresh and cryopreserved mitochondria 0.2 ml was added to 0.8 ml of incubation buffer at pH 7.6 consisting of 90 mM KC1 (Merck), 50 mM N-(Tris-(hydroxymethyl)methyl)glycine (Tricine) (Merck), 10 m&f MgCl, (Merck), 5 m&f potassium phosphate buffer (Merck), 1 mM EDTA (Calbiochem), and as energy source: 2 m&f ATP (Sigma, St. Louis, MO.), 5 m&f phosphoenolpyruvate (Sigma), and 10 pg/ml pyruvate kinase (Sigma). Each 100 ml of incubation buffer equally contained 1 ml of an L-amino acid supplement (Vamin, Vitrum, Stockholm 12, Sweden). To each final 1 ml of incubation medium containing the mitochondria 1 pCi of L-[U-‘4C]isoleucine (sp radioact: 300 mCi/mmol; Amersham International, Amersham, Bucks, UK) was added to follow the incorporation into proteins during incubation in a shaking waterbath at 25°C over a period of up to 60 min. To follow the amino acid uptake into the mitochondria as a measure of membrane transport activity, incu-
MITOCHONDRIAL
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bation with 1 pCi of L-[U-14Clisoleucine was performed in the presence of 200 pg/ml of chloramphenicol (Sigma), completely blocking protein synthesis (3, 22). In some experiments, the final mitochondrial suspensions were incubated in the presence of 0.6, 1.2, or 1.g M of supplementary addition of Me,SO. All media used were passed through a 0.22~p.m Nalgene disposable filter (Nalge Cy., Rochester, NY) before incubation was started to avoid the possibility of bacterial contamination. Radioisotope Evaluation After incubation of fresh and cryopreserved mitochondria with L-[U-14C]isoleucine, protein synthesis was arrested by adding 5 ml of trichloroacetic acid (TCA, Merck) 20%. After three more washes with 10 ml of TCA, the precipitated proteins were dissolved in 2 ml of Lumasolve (Lumac Systems AG, Basel, Switzerland) by heating overnight at 60°C. After the addition of 10 ml of Lipoluma (Lumac Systems AG), the incorporated radioactivity was counted in a liquid scintillation spectrometer (Rack Beta, LKB, Wallac, OY , Turku, Finland). For membrane transport experiments amino acid uptake was stopped after incubation by the addition of 10 ml of fresh icecold incubation medium containing 30 mmol of nonradioactive isoleucine (Sigma). After centrifugation at 10,OOOg for 10 min, followed by three identical washes with fresh isoleucine-containing medium, the precipitated mitochondria were dissolved in Lumasolve and were further processed to evaluate the accumulation of radiolabeled amino acid. Radioactive measurements (after subtraction of the zero time values serving as control blanks) were expressed as disintegrations per minute (DPM) per milligram of mitochondrial proteins 2 SEM. Statistical evaluation of significance was carried out according to Student’s t test.
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RESULTS
Effect of Me,SO on Protein Synthesis and Amino Acid Transport of Fresh Mitochondria The results of the experiments to study protein synthesis in response to Me,SO exposure are shown in Fig. 1. The incorporation of L-[U-‘4C]isoleucine into the proteins of fresh mitochondria, not previously exposed to Me,SO but equally treated by centrifugation and dilution as for the exposed
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FIG. 1. Effect of Me,SO on the incorporation of L[U-‘4C]isoleucine into the proteins of fresh mitochondria. Mitochondria were prepared as described under Materials and Methods. Mitochondriaf suspension (0.2 ml) was added to 0.8 ml of incubation medium and incubated at 25°C for up to 60 min in the presence of 1pCi L-[U-r4C]isoleucine (specific radioact: 300 mCi/ mmol) and of an energy source. Apart from the sample containing fresh mitochondria (0) Me,SO was added to the incubation samples in final concentrations of 0.6 (0), 1.2 (m), and 1.8 M (0). The amino acid incorporation into the mitochondrial proteins is expressed as disintegrations per min (DPM). Each value represents the average of six experiments k SEM.
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samples, progressed linearly over the incubation period of 60 min at 25°C. After exposure to 1.8 M Me,SO for 30 min, the washed mitochondria were pelleted, resuspended in an original volume of homogenization medium, and aliquoted 0.2 ml in 0.8 ml of incubation medium, resulting in a residual Me,SO concentration of less than 0.05 M. These exposed mitochondria did not show significantly different incorporation pattern from the fresh sample upon incubation. As increasing concentrations of Me,SO were added to the incubation buffer in which the mitochondria were suspended, incorporation of the amino acid progressively decreased. Linearity was still observed in the presence of 0.6 M Me,SO but after 60 min of incubation incorporation amounted to 75% (p < 0.001 compared to controls). In the presence of 1.2 and 1.8 M Me,SO the linear phase of incorporation lasted only 30 and 20 min, respectively, while after 60 min of incubation, compared to the samples not containing any additional Me,SO, the incorporated isoleucine amounted to 50% (P < 0.001) and 28% (P < O.OOl), respectively, with the differences becoming more marked as incubation progressed. In Fig. 2 similar studies on Me$O exposure as it affects membrane amino acid transport is shown. In the presence of chloramphenicol, blocking protein synthesis, membrane transport was evaluated by measuring the r,-[U-14C]isoleucine uptake by the mitochondria. Amino acid uptake by fresh mitochondria progressed linearly up to 10 min followed by a leveling off with only a slow increase up to 40 min. Without any addition of Me,SO to the incubation medium, but only with the residual Me,SO present after exposure to the cryoprotectant, amino acid uptake presented an identical pattern as observed with fresh mitochondria. After the addition of 1.8 M Me,SO to the incubation medium containing the mitochondria, a linear uptake con-
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lncubahon
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FIG. 2. Effect of Me,SO on the L-[U-‘4C]isoleucine uptake by fresh mitochondria. Mitochondria prepared as in Fig. 1 were incubated in the presence of 200 &ml chloramphenicol. After incubation for up to 40 min at 25”C, the control samples (0) and the samples containing a final concentration of 1.8 M Me,SO (0) received 10 ml of fresh incubation buffer containing 30 mmol of nonradioactive isoleucine by which label up take was arrested. After repeated washes the samples were prepared for radioactive counting. Each value represents the average uptake values of six experiments in DPM * SEM.
tinued for 10 min although only reaching 89% of the control samples and equally leveling off to a value of 80% after 40 min of incubation (P < 0.001). The uptake values in the presence of intermediate 0.6 and 1.2 M Me,SO concentrations all fell in between the two illustrated curves (data not shown). Effect of Cryopreservation on L-[ U-14C]isoleucine Incorporation and Uptake The various freezing regimes did not significantly affect the incorporation rates of L-[U-14C]isoleucine into the proteins of cryopreserved mitochondria during the initial 10 min of incubation. However as incu-
MITOCHONDRIAL
FUNCTION
bation progressed, fast cooling achieved by immediate submersion of the mitochondria into liquid nitrogen without the cryoprotection of Me,SO resulted in the recovery of 60% of incorporation activity. In contrast the presence of Me,SO during cooling only resulted in 30% of isoleucine incorporation compared to the nonfrozen samples evaluated after longer incubation (60 min) (P < 0.001). Using the slow cooling method in the presence of 1.8 M of Me,SO the isoleucine incorporation amounted to 40% of the fresh control mitochondria after 60 min of incubation while slow cooling without Me,SO resulted in 52% of amino acid incorporation. Without the cryoprotectant Me,SO, fast cooling already resulted in a significantly higher incorporation of isoleutine after 20 min of incubation, while in the presence of Me,SO slow cooling proved beneficiary when incubation was prolonged beyond 30 min (Fig. 3). The values in mitochondria were always higher in the absence of Me,SO (fast, P < 0.001; slow, P < 0.001) than when the cryoprotectant was present during cryopreservation. When membrane transport activity was examined after thawing, the cryopreserved mitochondria presented a linear isoleucine uptake in less than 10 min, achieving a maximal uptake which remained almost constant for up to 30 min of incubation (Fig. 4). When evaluating the L-[U-14C]isoleucine uptake, there were no obvious differences between fast- and slow-cooling rates in the absence of Me,SO. In both circumstances uptake values were significantly higher than that achieved by mitochondria exposed to Me,SO during cooling (P < 0.01 in each case). With Me,SO present during slow cooling, the uptake values during subsequent incubation appeared slightly higher. In samples frozen without Me,SO the L-[U-14C]isoleucine uptake after 30 min of incubation amounted to 28% of the fresh nonfrozen mitochondria, while freezing in the presence of Me,SO generally reduced
AFTER
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CRYOPRESERVATION
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3. Incorporation of L-[U-i4C]isoleucine into the proteins of fresh and cryopreserved mitochondria. Preparation, incubation, and processing of the fresh mitochondria (0) was carried out as for Fig. 1. Freezing took place in the presence of 1.8 M Me,SO following a fast cooling rate achieved by immediate submersion into liquid nitrogen (A) or by a slow cooling rate, initially 1°C per min up to -36°C followed by exposure for 30 min in the N, gasphase and subsequent submersion (A). Other samples were frozen without Me,SO either quickly (m) or slowly (0). Incubation was carried out after the removal of the cryoprotectant by dilution or after analogous dilution of the samples not being exposed to Me,SO. Each value represents the mean of six experiments ? SEM. FIG.
the amino acid uptake by a further 8 to 12% (Fig. 4). DISCUSSION
The in vitro amino acid incorporation into proteins by isolated mitochondria, dependent on enzyme systems involved in oxidative phosphorylation, is generally supported by an ATP-regenerating system (18, 26, 30). However, high-energy intermedi-
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FIG. 4. L-[U-“‘C]isoleucine uptake by fresh and cryopreserved mitochondria. Preparation and incubation of fresh mitochondria was carried out as for Fig. 2. Freezing and thawing was carried out as for Fig. 3. Apart from fresh nonfrozen samples (O), mitochondria were frozen in the presence of 1.8 M Me,SO according to a fast cooling (A) or a slow cooling (A) regimen. Samples to be frozen without Me,SO were subjected to either fast cooling (W) or slow cooling (0). Each value represents the mean of six experiments 2 SEM.
ates of oxidative phosphorylation generated by electron transport or ATP hydrolysis may not always be the exclusive source of energy for mitochondrial protein synthesis, as this complex mechanism appears to be governed by several energy-dependent processes (30). Analyzing the in vitro proteinsynthesizing activity, sometimes apparently conflicting observations have emerged concerning ideal conditions and circumstances in which these metabolic functions should be measured. Although ATP generated by respiration or transported across the membrane is essential for
AND
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optimal amino acid incorporation, the necessity or advisability of adding exogenous adenine nucleotides or ATP-regenerating systems has been queried (3, 21, 22). Furthermore, depending on the source of the mammalian mitochondria from different organs, or organelles originating from microorganisms, yeasts, fungi, or plants, the advised experimental conditions may greatly vary (3, 15, 16, 19, 22). As we intend to compare metabolic activities, without trying to improve experimental conditions or to explore the complex reactions of mitochondrial protein synthesis, a well-proven method has been basically followed (2). In all instances the incubation medium contains ATP and an ATP-regenerating system. Since the amino acid incorporation curves present an initial linear ATPdependent progression, and all solutions were passed through a 0.22 pm filter before use, bacterial growth interference may be excluded (30). As no transport mechanism for a-amino isobutyric acid has been demonstrated in isolated mitochondria, amino acid transport activity was evaluated in our study by observing L-[U-‘4C]isoleucine uptake after blocking the protein-synthesizing activity by chloramphenicol (4, 8). Amino acid uptake appears to reach maximal levels during the initial 10 min of incubation for fresh organelles. Although some authors report a rapid decline of intramitochondrialfree amino acid levels on continuing incubation, this observation has not always been confirmed (3,4, 12, 30). Blocking protein synthesis and allowing membrane transport to take place affect the efficiency and the general pattern of the transport mechanism. Initial accumulation of precursor amino acids may well induce a concentration-driven self-limiting transport activity. Addition of chloramphenicol to block protein synthesis considerably reduces the absolute values of transport activity presumably because the amino acids are not being consumed in the production of new
MITOCHONDRIAL
FUNCTION
AFTER CRYOPRESERVATION
451
polypeptide chains (8). The cryopreserved leads to morphological and functional modmitochondria particularly appear affected ifications, it still appears least damaging to by chloramphenicol and present persisthe protein-synthesizing activity. As memtantly lower uptake values than to be ex- brane transport and protein synthesis bepected from the protein synthesis expericome inhibited when incubation takes place ments. in the presence of Me,SO, these mitochonIndiscriminate repeated freezing and drial functions appear best preserved when thawing seriously damages mitochondrial freezing takes place in a medium without protein synthesizing activity thus reducing Me,SO. Furthermore it appears that withthe isoleucine incorporation (21, 25). Cryoout Me,SO present, the normal advantages preservation leads to partial loss of the cy- of a slow cooling rate disappear. In our systochrome C pool released from the inner tem ATP and an ATP-regenerating system mitochondrial membrane into the incubaare both provided to fresh and cryopretion medium through the damaged outer served mitochondria, so that cryoinjury to membrane. This enzyme depletion affected the protein synthesizing activity appears by different freeze-thaw regimes is at least primarily to occur at the levels of structure one of the causes of inhibited mitochondrial or membrane organization (9, 15, 23). electron transport chain activities (23). AlApart from cryoinjury some other forms though Me,SO provides protection to the of chemical toxicity inhibit oxidative phosmitochondrial substrate-linked oxygen con- phorylation and also increase mitochonsumption, sole exposure to and dilution of drial membrane permeability (6). In rat the cryoprotectant already results in some brain mitochondria chemical inhibition of changes of mitochondrial function (7, 11, protein synthesis does not directly affect 14, 21). Reduced efficiency of oxygen up- ATP synthesis. Cryoinjury appears to aftake is already observed at concentrations fect protein synthesis on a nonspecific basis of around 0.1 M of Me,SO while inhibitory as already observed in other conditions. effects on protein synthesis only appear at Chemically induced modifications in the much higher levels. As oxidative phosrate of mitochondrial protein synthesis rephorylation is much more sensitive to sult in a nonselective stimulatory or inhibicryoinjury this process shows evidence of tory effect on the protein fractions formed serious impairment long before ultrastruc(19). The stimulatory effects of cold exposure on in viva rat liver mitochondrial proture and protein metabolism are affected (9). Since Me,SO has been observed to tein synthesis by the mechanism of instimulate mitochondrial ATP production creased triiodothyronine levels equally (possibly by equilibrium shifts between in- take place without specifically affecting volved enzyme systems) the observed in- any of the major proteins derived from mihibitory effects are unlikely to be due to tochondrial DNA (16). For the present adequate cryopreservainterference at this bioenergetic level (27). Mitochondrial protein synthesis proves far tion of functional integrity of membrane activity more resistant to freeze-thaw injury than transport and protein-synthesizing in isolated mitochondria remains problemthat seen in the original intact hepatocyte from which they are derived (13). The pro- atic and appears to be optimally achieved tection of isolated mitochondria compared during fast cooling without the addition of to the mitochondria frozen in situ during an additional permeating cryoprotectant. cooling without Me,SO may well be due to ACKNOWLEDGMENTS the relatively high sucrose levels present during preparation and cryopreservation The authors are indebted to the Belgian National Foundation for Medical Research (F.G.W.O.) for a (14). Although fast freezing without Me,SO
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FULLER,
grant to the laboratory, to the Nationale Loterij for a research grant, and to Mrs. F.De Wever for her valuable technical assistance.
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13. Fuller, B. J., Grout, B. W., and Woods, R. J. Biochemical and ultrastructural examination of cryopreserved hepatocytes in rat. Cryobiology 19, 493-502
REFERENCES
1. Araki, T. Freezing injury in mitochondrial membranes. I. Susceptible components in the oxidation systems of frozen and thawed rabbit liver mitochondria. Cryobiology 14, 144-150 (1977). 2. Beattie, D. S. Yeast versus mammalian mitochondrial protein synthesis. In “Methods in Enzymology” (S. Fleischer and L. Packer, Eds.), Vol. 56, pp 17-27. Academic Press, New York, 1979. 3. Beattie, D. S., and Ibrahim, N. G. Optimal conditions for amino acid incorporation by isolated rat liver mitochondria: Stimulation by valinomycin and other agents. Biochemistry 12, 176 180 (1973). 4. Buchanan, J., Popovitch, J. R., and Tapley, D. F. Leucine transport by rat liver mitochondria in vitro. Biochim. Biophys. Acta 173, 532-539 (1969). 5. De Giorgi, C., and Siculella, L. Stimulation of protein synthesis in isolated mammalian mitochondria by a factor in the cytosol. Cell Biol. Znt. Rep. 8, 587-590 (1984). 6. De Wit, R. H., and Brabec, M. J. Protein synthesis by hepatic mitochondria isolated from carbon tetrachloride-exposed rats. Biochim. Biophys. Acra 824, 256-261 (1985). 7. Dickinson, D., Misch, M., and Drury, R. Dimethyl sulfoxide protects tightly coupled mitochondria from freezing damage. Science 156, 1738-1739 (1967). 8. Finzi, E., Clejan, L., and Beattie, D. S. Effect of temperature on protein synthesis and leucine transport by yeast mitochondria. Me&r. Biothem. 5, 291-307 (1985). 9. Fishbein, W. N., and Griffin, J. L. Studies on the mechanism of freezing damage to mouse liver. IV. Effects of ultrarapid freezing on structure and function of isolated mitochondria. Cryobiology 13,542-556(1976). 10. Fleischer, S. Long-term storage of mitochondria to preserve energy-linked functions. In “Methods in Enzymology” (S.Fleischer and L.Packer, Eds.), Vol. 55, pp. 28-32. Academic Press, New York, 1979. 11. Foissner, I. Septation and fragmentation in Oedogonium mitochondria as different and independent effects of dimethyl sulfoxide (DMSO) treatment. Experientia 42, 958-%0 (1986). 12. Freedland, R. A., Crozier, G. L., Hicks, B. L., and Meijer, A. J. Arginine uptake by isolated rat liver mitochondria. Biochim. Biophys. Acra 802,407--112
(1984).
(1982).
14.
Fuller, B. J., Rubinacci, A., Geboes, K., and De Loecker, W. The bioenergetics of mitochondria after cryopreservation. Cryobiology 26, 333-
15.
Gadaleta, M. N., Minervini, G. R., Renis, M., De Giorgi, C., and Giovine, A. Mitochondrial DNA, RNA and protein synthesis in normal and hypothyroid developing rat liver. Cell Differ. 19, 4349 (1986). Goglia F., Liverini, G., Lanni, A., and Barletta, A. Mitochondrial DNA, RNA and protein synthesis in normal, hypothyroid and mildly hyperthyroid rat liver during cold exposure. Mol. Cell. Endocrinol. 55, 141-147 (1988). Hay, R., Bohni, P., and Gasser, S. How mitochondria import proteins. Biochim. Biophys. Acta 779, 65-87 (1984). Kroon, A. M. Protein synthesis in mitochondria. II. A comparison of mitochondria from liver and heart with special reference to the role of oxidative phosphorylation. Biochim. Biophys. Acta 91, 145-154 (1964). Kuznetsov, D. A., and Musajev, P. I. 0. Chemical-induced modulation of ATP and protein synthesis processed inside rat brain mitochondria. ht. J. Neurosci. 38, 331-343 (1988). Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein measurement with the Fohn Phenol reagent. J. Biol. Chem. 193,
340 (1989).
16.
17.
18.
19.
20.
265-275(1951).
Mills, N. C., Durwood, B. R., Littlejohn, R. A., Horst, I. A., and Kowal, J. Optimalization of in vitro protein synthesis by isolated mouse adrenal mitochondria. Anal. Biochem. 138, l&180 (1984). 22. Mockel, J. J., and Beattie, D. S. Optimal conditions for studies of amino acid incorporation in vitro by isolated skeletal muscle mitochondria. Arch. Biochem. Biophys. 167, 301-310 (1975). 23. Petrenko, A. Y., and Subbota, N. Inhibition of the mitochondrial electron transport chain by low temperatures: Losses of cytochrome C. Cryo 21.
Lett.
7, 395-2
(1986).
Privitera, C. A., Greiff, D., Strength, D. R., Anglin M., and Pinkerton, H. Oxidative phosphorylation by mitochondrial suspensions after freezing and storage at low temperatures. J. Biol. Chem. 233, 526527 (1958). 25. Roodyn, D. B. Further study of factors affecting amino acid incorporation into protein of isolated mitochondria. Biochem. J. 97, 782-793 24.
(1965).
MITOCHONDRIAL
FUNCTION
26. Roodyn, D. B., Reis, P. J., and Work, T. S. Protein synthesis in mitochondria: Requirements for the incorporation of radioactive amino acids into mitochondrial protein. Biochem. J. 80, % 21 (l%l). 27. Sakamoto, J. Effect of dimethyl sulfoxide on ATP synthesis by mitochondrial soluble F,-ATPase. J. Biochem. 96, 483-487 (1984). 28. Sherman, J. K. Correlation in ultrastructural cryoinjury of mitochondria with aspects of their
AFTER CRYOPRESERVATION
453
respiratory function. Exp. Cell. Res. 66, 371 384 (1971). 29. Tsvetkov, T., Tsonev, L., Meranzov, N., and Minkov, I. Functional changes in mitochondrial properties as a result of their membrane cryodestruction. Cryobiology 22, 47-54 (1985). 30. Wheeldon, L. W., and Lehninger, A. L. Energylinked synthesis and decay of membrane proteins in isolated rat liver mitochondria. Biochemistry 5, 3533-3545 (1966).
