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Thyroxine-induced changes in rat liver mitochondrial cytochromes
Molecular and Cellular Endocrinology, 41 (1985) 163-169 Elsevier Scientific Publishers Ireland, Ltd.
163
MCE 01329
Thyroxine-induced
changes in rat liver mitochondrial
cytochromes *
Mark A. Horrum, Richard B. Tobin and Robert E. Ecklund Department of Internal Medicine, University of Nebraska Medical Center and Veterans Administration NE 68105 (U.S.A.)
Medical Center, Omaha,
(Received 25 February 1985; accepted 11 March 1985)
Keywords:
hyperthyroidism;
mitochondrial
respiration; cytochrome
redox state; loose coupling.
Summary
The effects of 10 days of thyroxine injection (15 pg/lOO g body weight) on rat liver mitochondrial cytochrome concentration and on the percent reduction of the individual cytochromes during succinatedriven state III and IV respiration was spectrophotometrically determined at cytochrome-specific wavelength pairs. The concentrations of cytochromes b, c, total c (c + c,) and a a3 increased in hyperthyroid rats. The concentration of cytochrome c, remained constant in euthyroid and hyperthyroid rats. Changes in the concentration of the membrane-bound cytochromes were also determined by difference spectra in cytochrome c-depleted mitochondrial membranes. Cytochromes b and a a3 showed increased concentrations in hyperthyroid rats while the concentration of cytochrome ci remained unchanged. Hyperthyroid mitochondria showed increased reduction of cytochromes b, c,, c and total c during state III respiration and cytochromes cl, c, and total c during state IV respiration. The percent reduction of cytochrome b decreased during state IV respiration in the hyperthyroid mitochondria. These results suggest that the increase in respiration observed in the hyperthyroid state may be related to changes both in the mitochondrial cytochrome concentration and in the cytochrome reduction level.
A major effect of thyroid hormones is to increase mitochondrial respiration. While this has been known for many years (Drabkin, 1949; Lardy and Feldott, 1951; Martius and Hess, 1951), how the increase is regulated is still a question. Early proposals suggested thyroid hormones act as uncouplers of respiration (Lardy and Feldott, 1951; Martius and Hess, 1951). But the uncoupling only occurred at high, toxic doses of thyroid hormones, * Supported in part by the Medical Research Service of the Veterans Administration and in part by the Bly Memorial Research Fund, University of Nebraska Medical Center. Send correspondence to: Dr. Mark A. Horrum, V.A. Medical Center, Research Service (R-217), 4101 Woolworth Ave., Omaha, NE 68105 (U.S.A.).
0303-7207/85/$03.30
not at physiological levels (Tata et al., 1963). Another proposal suggested the respiratory chain responds as a unit to the thyroid status with hyperthyroidism resulting in increased cytochrome concentration and hypothyroidism resulting in decreased concentrations (Roodyn et al., 1965; Nishiki et al., 1978). However, this does not appear to be the only mechanism capable of regulating respiration. Hypothyroid rats, treated with thyroid hormones, developed increased respiration in the absence of an enlargement of the cytochrome content (Tata et al., 1963; Roodyn et al., 1965; Bronk, 1966; Sterling et al., 1980). This increase in respiration was related to an increased reduction of the cytochromes due to an enhanced
0 1985 Elsevier Scientific Publishers Ireland, Ltd.
164
rate of cytochrome reduction (Bronk, 1966). Thus, thyroid hormones may affect respiration by at least 2 ways, increasing the content of cytochromes and/or altering the rate of reduction of the cytochromes. In order to gain a better understanding of the effects of thyroid hormone on the regulation of respiration, we have determined the hormone-induced changes in individual cytochrome concentrations and in their percent reduction during states III and IV respiration. We find that hyperthyroidism results in an increase in the concentration of some, but not all, of the cytochromes. We also find differences in the percent reduction of the individual cytochromes during respiration. A preliminary report of this data was presented at the 1983 FASEB meetings (Horrum and Tobin, 1983). Materials and methods Treatment of rats Male Sprague-Dawley rats (Charles River, Wilmington, MA) initially weighing 175-250 g were used throughout this study. The animals were given laboratory chow and water ad libitum and maintained in a temperature and humidity controlled room with equal 12 h periods of light and dark. Rats were made hyperthyroid by subcutaneous daily injections, for 10 days, of thyroxine (T4) at 15 pg T,/lOO g body weight. The thyroxine was dissolved in 50 mM NaOH at a concentration of 150 pg T,/ml of NaOH. Control rats were injected daily for 10 days with 50 mM NaOH. 24 h after the last injection, the rats were killed by decapitation, the livers were excised quickly and cooled in 250 mM sucrose, 1 mM Tris HCl, pH 7.4. Blood was collected from the neck to determine serum Td levels. Isolation and treatment of mitochondria Liver mitochondria used for the determination of the cytochrome reduction state were prepared according to the procedure of Johnson and Lardy (1967) except the mitochondria were washed and resuspended a total of 4 times. Oxygen consumption was measured polarographically at 25°C as described previously (Tobin et al., 1972), or in some experiments with a vibrating platinum electrode (American Instrument Co., Silver Springs,
MD) during simultaneous spectrophotometric analysis of the cytochromes. Oxygen consumption and spectrophotometric analysis were done in medium as described by Tobin et al. (1972). Mitochondria used for the cytochrome spectra were isotonically isolated by the method of Siess (1983). Cytochromes c-depleted mitochondrial membranes were prepared by repetitively freezethawing the mitochondria in 0.9% KC1 (Williams, 1964). Enzyme activity a-Glycerophosphate dehydrogenase activity was determined by the method of Wernette et al. (1981). Succinate dehydrogenase was performed according to the method of Ackrell et al. (1978). Determination of reduction state of the cytochromes The percent reduction of the cytochromes was determined at 25°C using the dual-wavelength mode of an Aminco DW-2a spectrophotometer (American Instrument Co., Silver Springs, MD). The following cytochrome-specific wavelength pairs were used throughout the experiment: Total cytochrome c, 550-540 nm (Chance and Williams, 1956); c, 550-535 nm; ci, 554-540 nm; b, 563-577 nm; a a3, 605-630 nm (Vanneste, 1965). Cytochrome percent reduction was determined in a final volume of 3.0 ml by adding 2.5 mg of the isolated mitochondria to the medium in a cuvette with magnetic stirring to prevent sedimentation. After the initial spectrophotometric reading stabilized, rotenone (2 PM) was added and the resulting decrease in absorbance was taken as corresponding to 100% oxidation of the cytochrome. In some experiments 1 PM carbonyl cyanide m-chlorophenylhydrazone (CCCP), an uncoupler, was added to ensure complete oxidation. However, the addition of CCCP to rotenone-blocked mitochondria resulted in very little further oxidation (data not shown). Since CCCP would prevent determination of the percent reduction during states III and IV respiration, the uncoupler was not routinely utilized. To the oxidized mitochondria, 4.5 mM succinate was added and the increase in reduction allowed to stabilize. This was followed by the addition of 400 nmol of ADP with the resulting stabilized decrease in absorbance taken as the state III respiratory level. Absorbance then
165
returned to the pre-ADP level and this level was calculated as the state IV respiratory level. The cytochromes were then reduced by the addition of 2 mM KCN. This level was taken as 100% reduced. The cytochrome percent reduction was calculated as: AOD ((state III or IV level - 100) oxidized level) AOD (100% reduced level - 100% oxidized level) x 100. The percent reduction for each wavelength pair was performed in triplicate for each sample.
The concentration of the cytochromes in intact mitochondria was estimated by the AOD between the 100% reduced level and the 100% oxidized level at the appropriate wavelength pairs, using the extinction coefficient of Vanneste (1965). The content of total cytochrome c was determined from the absorbance difference at 550-540 nm as described previously (Nishiki et al., 1978). The relative concentration of membrane-bound cytochromes in cytochrome c-depleted membranes was determined by difference spectra according to Williams (1964). Other assays Serum T4 levels were determined with a Quantaphase Thyroxine Radioimmunoassay (Bio-Rad Laboratories, Richmond, CA). Protein was determined by the dye binding assay of Bradford (1976) with bovine serum albumin as the standard. Biochemical reagents used in this work were obtained from Sigma Chemical Co. (St. Louis, MO). The other chemicals were of the highest purity available. Statistical significances were determined by Student’s t-test. Results
The daily injection for 10 days of rats with 15 pg T,/lOO g body weight increased the T4 serum concentration, the specific activity of LTglycerophosphate dehydrogenase, and the rate of succinate respiration during states III and IV (Table 1). Serum T4 levels increased 2.5 to 3-fold in
TABLE 1 THYROXINE-INDUCED CHANGES IN SERUM T4 LEVELS, THE SPECIFIC ACTIVITIES OF aGLYCEROPHOSPHATE DEHYDROGENASE AND SUCCINATE DE~YDR~ENASE AND MIT~HONDRIAL RESPIRATION OF SUCCINATE Assays performed Serum T4 (cg Td/dl)
Euthyroid (II =lO) 5.8&- 0.3
a-glycerophosphate dehydrogenase nmol INT reduced 4.9* min/mg protein Succinate dehydrogenase pmol DCPIP reduced min/mg protein State III nmol0, min/mg mito protein State IV nmol0, m.in/mg mito protein Respiratory control ratio State III/State IV
0.5
Hyperthyroid (n=S) 15.5 f
1.3 **
2S.4*
1.2 **
407.2 f 27.3
513.0*39.s **
84.7* 5.6
103.8 * 7.1 **
l&5*
2.1
4.6& 0.3
25.84 1.3 **
4.0* 0.3 *
Values represent the mean f standard error. * = P -=z0.05. ** = P < 0.001. n = Number of animals per group.
the h~erthyroid rats compared to control rats. The specific activity of the thyroid hormone-induced enzyme, a-glycerophosphate dehydrogenase, increased 5- to 7-fold. The specific activity of succinate dehydrogenase increased by 25%. Oxygen consumption with succinate increased by approximately 20% during state III and by approximately 40% during state IV resulting in the decrease in the respiratory control ratio. The data presented in Table 1 demonstrates that the T,-injetted animals were hyperthyroid. Fig. 1 shows an example of the changes in absorption observed at 550-540 nm during succinate respiration. The addition of rotenone to the mitochondrial suspension caused essentially complete oxidation of total cytochrome c seen as a decrease in absorbance. The addition of succinate resulted in an increase in absorbance signifying the reduction of the cytochrome. The addition of
166
Fig. 1. Changes in the absorbance at 550-540 nm used to determine the concentration and percent reduction of total cytochrome c. Isolated mitochondria (2.5 mg protein) were studied at 25°C in an Aminco DWZA spectrophotometer. The abscissa is time in seconds. Downward deflection indicates an oxidation of the cytochromes, upward deflection designates reduction.
400 nmol of ADP resulted in the transient oxidation of the cytochrome (state III) followed by reduction back to the pre-ADP level (state IV). Alternating the concentration of ADP had no effect on the resulting reduction state but proportionally altered the time spent at the state III redox state. The cytochromes were then reduced by the addition of KCN. At all wavelength pairs and in both euthyroid and hyperthyroid mitochondria, the percent reduction of each cytochrome during states III and IV was mitochondrial protein-independent while the changes in absorption were directly proportional to protein concentration (data not shown). Hyperthyroidism resulted in large increases in TABLE
2
CYTOCHROME CONCENTRATIONS CHONDRIA ISOLATED FROM THE EUTHYROID OR HYPERTHYROID RATS Cytochrome
b Cl
c Total c a 03
the concentration of some, but not all, of the mitochondrial cytochromes. Table 2 shows that cytochromes 6, c, total c and a a3 increased in content in mitochondria isolated from hyperthyroid rats. The concentration of cytochrome c, in the hyperthyroid rat mitochondria was not significantly different from that of euthyroid rat mitochondria. A comparison of the concentrations of total cytochrome c and the sum of cytochromes ci and c found no significant difference between them (data not shown). Thus, the increase in concentration of total cytochrome c is due to an increase in the concentration of cytochrome c and not of cytochrome c,. The determination of cytochrome concentration by the above manner has several possible problems. The major potential errors are the overlaps in the spectra of cytochromes c and c, and the use of succinate and KCN to reduce the cytochromes. To resolve these difficulties, mitochondria were isolated and underwent exhaustive extraction of cytochrome c by 5 cycles of freeze-thawing the mitochondria in 0.9% KCl. Extraction of cytochrome c from these membranes was completed by the 5th cycle (data not shown). Difference spectra, using sodium dithionite as the reducing agent, were then performed on the cytochrome c-depleted membranes. Table 3 shows that cytochromes b and a a3 increased in concentration in hyperthyroid rats while the concentration of cytochrome c, remained constant. These data demon-
Concentration (nmol cyt/mg
IN MITOLIVERS OF
mito protein)
Euthyroid (n =lO)
Hyperthyroid (n=8)
0.09f0.01 0.17 f 0.02 0.18f0.01 0.32 f 0.02 0.29f0.03
0.16 fO.O1 0.18 f 0.02 0.29 f 0.03 0.46*0.03 0.54 f 0.04
Values represent the mean f standard NS = not significantly different. * =P
error.
TABLE
3
EFFECTS OF THYROXINE SPECIFIC CYTOCHROMES PLETED MITOCHONDRIAL Cytochrome
* NS * * *
b Cl
a
a3
Cytochrome
ON THE CONTENT OF IN CYTOCHROME c-DEMEMBRANES content
a
Euthyroid (n=9)
Hyperthyroid (n=7)
0.81 f 0.20 1.67kO.11 1.45 *0.12
1.56kO.20 1.79k0.17 2.52k0.13
* NS *
Values represent the mean f standard error. NS = Not significantly different. * =P
pairs
161 TABLE
4
THYROXINE-INDUCED CHANGES IN THE PERCENT REDUCTION OF THE MITOCHONDRIAL CYTOCHROMES DURING STATE III AND IV RESPIRATION WITH SUCCINATE Cytochromes
b cl c Total c a3
Percent reduction State IV
State III Euthyroid (n=13)
Hyperthyroid (n=17)
Euthyroid (n =13)
Hyperthyroid (n =17)
19.0f0.9 19.1k1.9 19.3kl.l
23.4i-0.9 24.8k0.9 25.7kO.8
* * *
19.1f1.2 13.0f0.9
26.2f0.8 14.71t0.9
* NS
61.7kO.9 30.2kO.7 28.3kl.l 30.7f0.8 22.9jcO.7
49.5k0.7 37.9k0.6 38.9f0.6 40.9rtO.7 23.9f0.7
Values represent the meanf standard NS = Not significantly different. * =P<0.001. n = Number of animals per group.
* * * * NS
error.
strate that the mitochondrial cytochrome chain does not respond as a structural unit in the hyperthyroid state. Mitochondria isolated from hyperthyroid rats not only showed changes in oxygen consumption (Table 1) and in cytochrome content (Tables 2 and 3), but also in the percent reduction of the mitochondrial cytochromes. Table 4 shows the percent reduction of the cytochromes during state III and IV respiration. During state III respiration cytochromes b, cl, c and total c were more reduced by succinate in the hyperthyroid mitochondria, while there was no significant change in the percent reduction of cytochrome a a3. During state IV respiration cytochromes c,, c and total c were also more reduced in hyperthyroid mitochondria. But, in contrast to the increase seen in state III respiration, the percent reduction of cytochrome b decreased during state IV respiration. Thus, hyperthyroidism not only results in changes in the concentration of the cytochromes but also in their reduction state. Discussion The increase in respiration observed in mitochondria isolated from hyperthyroid rats is well known (Drabkin, 1949; Lardy and Feldott, 1951; Martius and Hess, 1951). Thyroid status not
only regulates respiration, but also controls the concentration of electron transport components, such as ubiquinone (Edwin et al., 1960), the cytochromes (Drabkin, 1949; Edwin et al., 1960; Roodyn et al., 1965; Booth and Holloszy, 1975; Winder and Holloszy, 1977; Nishiki et al., 1978) and associated enzymes such as the atractylosidesensitive adenine nucleotide carrier (Babior et al., 1973; Portnay et al., 1973). Information of this sort led Nishiki et al. (1978) to propose that the components of the respiratory chain respond as a respiratory unit to the animals’ thyroid status. However, increased respiration can also occur in the absence of changes in cytochrome content but in the presence of more reduced cytochromes (Bronk, 1966). In order to determine the possible roles of these processes on the hormone-induced increased respiration, we have determined the concentration of each cytochrome and it’s redox state during state III and IV respiration. Hypothyroid rats have decreased concentration of cytochromes b, c and a a3 which increase with thyroid hormone replacement, lowered respiratory rates, and less reduced cytochrome states (Drabkin, 1949; Edwin et al., 1960; Roodyn et al., 1965; Bronk, 1966; Nishiki et al., 1978; Sterling et al., 1980). Hyperthyroid rats have elevated concentrations of cytochromes b, c and a a3 (Table 2; Drabkin, 1949; Edwin et al., 1960; Booth and Holloszy, 1975; Winder and Holloszy, 1977; Nishiki et al., 1978). These data would tend to support the regulation of cytochrome concentration by thyroid hormones through a unit response. But, in these studies, cytochrome c was measured either as total cytochrome c + c, at 550-540 nm (Drabkin, 1949; Edwin et al., 1960; Roodyn et al., 1965; Bronk, 1966; Nishiki et al., 1978) or as isolated cytochrome c (Booth and Holloszy, 1975; Winder and Holloszy, 1977). We find (Tables 2 and 3) that the concentration of cytochrome c, is unaltered by T., treatment and that the increase in cytochrome c + c, is due to an increase in the concentration of cytochrome c and not of cytochrome c,. This differential response of ci has also recently been demonstrated in mitochondria isolated from rat hepatocyte cultures treated with triiodothyronine (Tj) (Wilson and McMurray, 1983). They found elevated concentrations of cytochrome c, b and a a3 while the concentration of
168
ci remained unchanged. Thus, it appears that the regulation of cytochrome c, concentration differs from the regulation of the other cytochromes in hyperthyroidism. The increased oxidative capabilities of mitochondria isolated from hyperthyroid rats cannot entirely be due to increased respiratory units, as has been suggested (Nishiki et al., 1978) since cytochrome c, concentration remained constant. The rate of respiration is also related to the reduction state of the cytochromes. Within 3 h after T3 treatment of hypothyroid rats, mitochondrial respiration increased, cytochrome concentrations remained constant, and cytochromes b, c + cl and a a3 became more reduced (Bronk, 1966). 48 h after T3 treatment, respiration was further elevated (Tata et al., 1963; Bronk, 1966), cytochrome concentration remained virtually unchanged (Roodyn et al., 1965; Bronk, 1966) and all of the cytochromes, except b, continued to become more reduced (Bronk, 1966). These data led Bronk (1966) to propose that these increases in oxidative activity were not due to changes in the concentration of electron transport components but rather appeared due to an effect of Ts on the rate of reduction of the cytochromes. The relationship between increased respiration and an increased redox state of the cytochromes has also recently been observed for glucagontreated rats (Halestrap, 1982). Mitochondria isolated from the livers of glucagon-treated rats showed enhanced succinate respiration. Cytochrome spectra performed on these mitochondria demonstrated increased reduction of cytochromes b, cl and c during state III and state IV respiration. This enhanced reduction appears due to increased substrate availability for mitochondrial respiration (Kimura et al., 1984). How the hyperthyroid electron transport chain responds to the increased rate of cytochrome reduction apparently depends on the metabolic state of the mitochondria. We found in state III respiration, when respiration is limited by substrate oxidation (Chance, 1965) that cytochromes b, cl, c and c + ci were more reduced. This increased redox state appears due to enhanced electron flow from the site of substrate oxidation. A major controlling step for electron transport is at the site of pyridine nucleotide oxidation (Chance, 1965). In
hyperthyroid rat hearts, the mitochondrial [NAD]/[NADH] was elevated compared to control hearts, suggesting augmented electron flow from NADH (Nishiki et al., 1978). In our experiments, electrons from NADH were blocked by rotenone but the increased activity of succinate dehydrogenase indicates increased electron flow from this substrate. Succinate dehydrogenase activity increased in the T,-treated animals by approximately 27% while cytochromes b, cl and c were approximately 27% more reduced. Our data indicates that an equilibrium exists between the site of substrate oxidation and the redox states of cytochromes b, c and c, during state III in the hyperthyroids. During state IV respiration, when respiration is controlled by ADP availability (Chance, 1965) the relative oxidation of cytochrome b indicates the electron transport chain is in disequilibrium in the T,-treated animals. The apparent cause of this disequilibrium may be ‘loose coupling’ as suggested by Hassinen et al. (1971). These investigators found evidence for ‘loose coupling’ of the electron transport chain near cytochrome b in intact livers from hyperthyroid rats, wherein there was no inhibition of respiration by fructose and there was no cross-over point between the flavoproteins, indicators of the redox state of the nicotinamide nucleotides, and cytochrome b. Our data is also consistent with this concept of loose coupling. If loose coupling occurs it should be most apparent during state IV. We find that cytochrome b is more oxidized in state IV, as would be expected in ‘loose coupling’. We also find the relative changes in the rates of rnitochondrial respiration are greater during state IV respiration (Table 1). The rate of state IV respiration in the hyperthyroids was nearly 40% greater than the euthyroid rate, while the rate of state III respiration was increased by approximately 20% in the hyperthyroid mitochondria. These 2 observations support the concept that T4 treatment results in ‘loose coupling’ and this alteration occurs at or near cytochrome b. The disequilibrium of the electron transport chain during state IV respiration in the hyperthyroids is also seen in the more reduced redox state of cytochrome ci in contrast to the relative oxidation of b. The mechanism responsible for this
169
equilibrium remains unclear but may involve the transfer of electrons from an increased content of cytochrome b to an unchanged content of c,. The result of these T4-induced changes in the concentration and the redox states of the cytochromes is to increase respiration capacity. T4 treatment increases respiration through several mechanisms. One mechanism may be via loose coupling of the chain near cytochrome b. This would increase respiration when substrates were plentiful but ADP was not. Another mechanism is increasing the activity of cytochrome oxidase. We find that T4 induces an increased concentration of cytochrome a u3, components of cytochrome oxidase (Tables 2 and 3) and a more reduced redox state for cytochrome c, a rate-listing step for cytochrome oxidase (Nishiki et al., 1978). These thyroxine-induced changes could result in increased oxidase activity and increased respiration, especially when neither substrate nor ADP is limiting. Direct in vivo evidence for increased ATP synthesis is lacking. However, Tata et al. (1963) have shown that physiological doses of T4 produce enhanced metabolic rates and ~tochond~al respiratory rates without uncoupling of oxidative phosphorylation. Loose coupling and enhanced respiratory capability are potential mechanisms for thyrotoxic mitochondria to meet the increased energy demands of the whole animal. References Ackrell, B.A.C., Keamey, E.B. and Singer, T.P. (1978) Methods Enzymol. 53, 446-482. Babior, B.M., Creagan, S., Ingbar, S.H. and Kipnes, R.S. (1973) Proc. Natl. Acad. Sci. (U.S.A.) 70, 98-102. Booth, F.W. and Holloszy, J.O. (1975) Arch. Biochem. Biophys. 167, 674-677. Bradford, M. (1976) Analyt. Biochem. 72, 248-254. Bronk, J.R. (1966) Science 153. 638-639. Chance, B. (1965) In: Control of Energy Metabolism, Eds.: B.
Chance, R.W. Estabrook and J.R. WiIli~~n (Academic Press, New York) pp. 415-435. Chance, B. and Williams, G.R. (1956) In: Advances in Enzymology, Vol. 17, Ed: F.F. Nord (Interscience Publishers, New York) pp. 65-134. Drabkin, D.L. (1949) J. Biol. Chem. 182, 335-349. Edwin, E.E., Green, J., Diplock, A.T. and Bunyan, J. (1960) Nature (Lond.) 186, 725. Erecinska, M. and Wilson, D.F. (1982) J. Membr. Biol. 70, l-14. Halestrap, A.P. (1982) Biochem. J. 204, 37-47. Hassinen, I.E., Ylikahri, R.H. and Kahonen, M.T. (1971) Arch. Biochem. Biophys. 147, 255-261. Horrum, M.A. and Tobin, R.B. (1983) Fed. Proc. 42, 325. Johnson, D. and Lardy, H.A. (1967) Methods Enzymol. 10, 94-95. Kimura, S., Suzaki, T., Kobyashi, S., Abe, K. and Ogata, E. (1984) Biochem. Biophys. Res. Commun. 119, 212-219. Lardy, H.A. and Feldott, G. (1951) Ann, N.Y. Acad. Sci. 54, 531-536. Martius, C. and Hess, B. (1951) Arch. Biochem. Biophys. 33, 486-487. Nishiki, K., Erecinska, M., Wilson, D.F. and Cooper, S. (1978) Am. J. Physiol. 235, &212-C219. Portnay, G.I., McClendon, F.D., Bush, J.E., Braverman, L.E. and Babior, B.M. (1973) Biochem. Biophys. Res. Commun. 55,17-20. Roodyn, D.B., Freeman, K.B. and Tata, J.R. (1965) Biochem. J. 94, 628-641. Siess, E.A. (1983) Hoppe-Seylers Z. Physiol. Chem. 364, 279-290. Sterling, K., Brenner, M.A. and Sakurda. T. (1980) Science 210. 340-342. Tata, J.R., Emster, L., Lindberg, O., Arrhenius, E., Pedersen, S. and Hedman, R. (1963) Biochem. J. 86, 408-428. Tobin, R.B., Mackerer, CR. and Mehlman, M.A. (1972) Am. J. Physiol. 223, 83-88. Vanneste, W.H. (1965) Biochim. Biophys. Acta 113, 175-178. Wemette, M.E., Ochs, R.S. and Lardy, H.A. (1981) J. Biol. Chem. 256, 12767-12771. Williams, J.N. (1964) Arch. Biochem. Biophys. 107, 537-543. Wilson, E.J. and McMurray, W.C. (1983) Can. J. Biochem. Cell Biol. 61, 636-643. Wilson, D.F., Owen, C.S. and Holian, A. (1977) Arch. Biothem. Biophys. 167,749-762. Winder, W.W. and HoIIoszy, J.O. (1977) Am. J. Physid. 232, C18OX184.
163
MCE 01329
Thyroxine-induced
changes in rat liver mitochondrial
cytochromes *
Mark A. Horrum, Richard B. Tobin and Robert E. Ecklund Department of Internal Medicine, University of Nebraska Medical Center and Veterans Administration NE 68105 (U.S.A.)
Medical Center, Omaha,
(Received 25 February 1985; accepted 11 March 1985)
Keywords:
hyperthyroidism;
mitochondrial
respiration; cytochrome
redox state; loose coupling.
Summary
The effects of 10 days of thyroxine injection (15 pg/lOO g body weight) on rat liver mitochondrial cytochrome concentration and on the percent reduction of the individual cytochromes during succinatedriven state III and IV respiration was spectrophotometrically determined at cytochrome-specific wavelength pairs. The concentrations of cytochromes b, c, total c (c + c,) and a a3 increased in hyperthyroid rats. The concentration of cytochrome c, remained constant in euthyroid and hyperthyroid rats. Changes in the concentration of the membrane-bound cytochromes were also determined by difference spectra in cytochrome c-depleted mitochondrial membranes. Cytochromes b and a a3 showed increased concentrations in hyperthyroid rats while the concentration of cytochrome ci remained unchanged. Hyperthyroid mitochondria showed increased reduction of cytochromes b, c,, c and total c during state III respiration and cytochromes cl, c, and total c during state IV respiration. The percent reduction of cytochrome b decreased during state IV respiration in the hyperthyroid mitochondria. These results suggest that the increase in respiration observed in the hyperthyroid state may be related to changes both in the mitochondrial cytochrome concentration and in the cytochrome reduction level.
A major effect of thyroid hormones is to increase mitochondrial respiration. While this has been known for many years (Drabkin, 1949; Lardy and Feldott, 1951; Martius and Hess, 1951), how the increase is regulated is still a question. Early proposals suggested thyroid hormones act as uncouplers of respiration (Lardy and Feldott, 1951; Martius and Hess, 1951). But the uncoupling only occurred at high, toxic doses of thyroid hormones, * Supported in part by the Medical Research Service of the Veterans Administration and in part by the Bly Memorial Research Fund, University of Nebraska Medical Center. Send correspondence to: Dr. Mark A. Horrum, V.A. Medical Center, Research Service (R-217), 4101 Woolworth Ave., Omaha, NE 68105 (U.S.A.).
0303-7207/85/$03.30
not at physiological levels (Tata et al., 1963). Another proposal suggested the respiratory chain responds as a unit to the thyroid status with hyperthyroidism resulting in increased cytochrome concentration and hypothyroidism resulting in decreased concentrations (Roodyn et al., 1965; Nishiki et al., 1978). However, this does not appear to be the only mechanism capable of regulating respiration. Hypothyroid rats, treated with thyroid hormones, developed increased respiration in the absence of an enlargement of the cytochrome content (Tata et al., 1963; Roodyn et al., 1965; Bronk, 1966; Sterling et al., 1980). This increase in respiration was related to an increased reduction of the cytochromes due to an enhanced
0 1985 Elsevier Scientific Publishers Ireland, Ltd.
164
rate of cytochrome reduction (Bronk, 1966). Thus, thyroid hormones may affect respiration by at least 2 ways, increasing the content of cytochromes and/or altering the rate of reduction of the cytochromes. In order to gain a better understanding of the effects of thyroid hormone on the regulation of respiration, we have determined the hormone-induced changes in individual cytochrome concentrations and in their percent reduction during states III and IV respiration. We find that hyperthyroidism results in an increase in the concentration of some, but not all, of the cytochromes. We also find differences in the percent reduction of the individual cytochromes during respiration. A preliminary report of this data was presented at the 1983 FASEB meetings (Horrum and Tobin, 1983). Materials and methods Treatment of rats Male Sprague-Dawley rats (Charles River, Wilmington, MA) initially weighing 175-250 g were used throughout this study. The animals were given laboratory chow and water ad libitum and maintained in a temperature and humidity controlled room with equal 12 h periods of light and dark. Rats were made hyperthyroid by subcutaneous daily injections, for 10 days, of thyroxine (T4) at 15 pg T,/lOO g body weight. The thyroxine was dissolved in 50 mM NaOH at a concentration of 150 pg T,/ml of NaOH. Control rats were injected daily for 10 days with 50 mM NaOH. 24 h after the last injection, the rats were killed by decapitation, the livers were excised quickly and cooled in 250 mM sucrose, 1 mM Tris HCl, pH 7.4. Blood was collected from the neck to determine serum Td levels. Isolation and treatment of mitochondria Liver mitochondria used for the determination of the cytochrome reduction state were prepared according to the procedure of Johnson and Lardy (1967) except the mitochondria were washed and resuspended a total of 4 times. Oxygen consumption was measured polarographically at 25°C as described previously (Tobin et al., 1972), or in some experiments with a vibrating platinum electrode (American Instrument Co., Silver Springs,
MD) during simultaneous spectrophotometric analysis of the cytochromes. Oxygen consumption and spectrophotometric analysis were done in medium as described by Tobin et al. (1972). Mitochondria used for the cytochrome spectra were isotonically isolated by the method of Siess (1983). Cytochromes c-depleted mitochondrial membranes were prepared by repetitively freezethawing the mitochondria in 0.9% KC1 (Williams, 1964). Enzyme activity a-Glycerophosphate dehydrogenase activity was determined by the method of Wernette et al. (1981). Succinate dehydrogenase was performed according to the method of Ackrell et al. (1978). Determination of reduction state of the cytochromes The percent reduction of the cytochromes was determined at 25°C using the dual-wavelength mode of an Aminco DW-2a spectrophotometer (American Instrument Co., Silver Springs, MD). The following cytochrome-specific wavelength pairs were used throughout the experiment: Total cytochrome c, 550-540 nm (Chance and Williams, 1956); c, 550-535 nm; ci, 554-540 nm; b, 563-577 nm; a a3, 605-630 nm (Vanneste, 1965). Cytochrome percent reduction was determined in a final volume of 3.0 ml by adding 2.5 mg of the isolated mitochondria to the medium in a cuvette with magnetic stirring to prevent sedimentation. After the initial spectrophotometric reading stabilized, rotenone (2 PM) was added and the resulting decrease in absorbance was taken as corresponding to 100% oxidation of the cytochrome. In some experiments 1 PM carbonyl cyanide m-chlorophenylhydrazone (CCCP), an uncoupler, was added to ensure complete oxidation. However, the addition of CCCP to rotenone-blocked mitochondria resulted in very little further oxidation (data not shown). Since CCCP would prevent determination of the percent reduction during states III and IV respiration, the uncoupler was not routinely utilized. To the oxidized mitochondria, 4.5 mM succinate was added and the increase in reduction allowed to stabilize. This was followed by the addition of 400 nmol of ADP with the resulting stabilized decrease in absorbance taken as the state III respiratory level. Absorbance then
165
returned to the pre-ADP level and this level was calculated as the state IV respiratory level. The cytochromes were then reduced by the addition of 2 mM KCN. This level was taken as 100% reduced. The cytochrome percent reduction was calculated as: AOD ((state III or IV level - 100) oxidized level) AOD (100% reduced level - 100% oxidized level) x 100. The percent reduction for each wavelength pair was performed in triplicate for each sample.
The concentration of the cytochromes in intact mitochondria was estimated by the AOD between the 100% reduced level and the 100% oxidized level at the appropriate wavelength pairs, using the extinction coefficient of Vanneste (1965). The content of total cytochrome c was determined from the absorbance difference at 550-540 nm as described previously (Nishiki et al., 1978). The relative concentration of membrane-bound cytochromes in cytochrome c-depleted membranes was determined by difference spectra according to Williams (1964). Other assays Serum T4 levels were determined with a Quantaphase Thyroxine Radioimmunoassay (Bio-Rad Laboratories, Richmond, CA). Protein was determined by the dye binding assay of Bradford (1976) with bovine serum albumin as the standard. Biochemical reagents used in this work were obtained from Sigma Chemical Co. (St. Louis, MO). The other chemicals were of the highest purity available. Statistical significances were determined by Student’s t-test. Results
The daily injection for 10 days of rats with 15 pg T,/lOO g body weight increased the T4 serum concentration, the specific activity of LTglycerophosphate dehydrogenase, and the rate of succinate respiration during states III and IV (Table 1). Serum T4 levels increased 2.5 to 3-fold in
TABLE 1 THYROXINE-INDUCED CHANGES IN SERUM T4 LEVELS, THE SPECIFIC ACTIVITIES OF aGLYCEROPHOSPHATE DEHYDROGENASE AND SUCCINATE DE~YDR~ENASE AND MIT~HONDRIAL RESPIRATION OF SUCCINATE Assays performed Serum T4 (cg Td/dl)
Euthyroid (II =lO) 5.8&- 0.3
a-glycerophosphate dehydrogenase nmol INT reduced 4.9* min/mg protein Succinate dehydrogenase pmol DCPIP reduced min/mg protein State III nmol0, min/mg mito protein State IV nmol0, m.in/mg mito protein Respiratory control ratio State III/State IV
0.5
Hyperthyroid (n=S) 15.5 f
1.3 **
2S.4*
1.2 **
407.2 f 27.3
513.0*39.s **
84.7* 5.6
103.8 * 7.1 **
l&5*
2.1
4.6& 0.3
25.84 1.3 **
4.0* 0.3 *
Values represent the mean f standard error. * = P -=z0.05. ** = P < 0.001. n = Number of animals per group.
the h~erthyroid rats compared to control rats. The specific activity of the thyroid hormone-induced enzyme, a-glycerophosphate dehydrogenase, increased 5- to 7-fold. The specific activity of succinate dehydrogenase increased by 25%. Oxygen consumption with succinate increased by approximately 20% during state III and by approximately 40% during state IV resulting in the decrease in the respiratory control ratio. The data presented in Table 1 demonstrates that the T,-injetted animals were hyperthyroid. Fig. 1 shows an example of the changes in absorption observed at 550-540 nm during succinate respiration. The addition of rotenone to the mitochondrial suspension caused essentially complete oxidation of total cytochrome c seen as a decrease in absorbance. The addition of succinate resulted in an increase in absorbance signifying the reduction of the cytochrome. The addition of
166
Fig. 1. Changes in the absorbance at 550-540 nm used to determine the concentration and percent reduction of total cytochrome c. Isolated mitochondria (2.5 mg protein) were studied at 25°C in an Aminco DWZA spectrophotometer. The abscissa is time in seconds. Downward deflection indicates an oxidation of the cytochromes, upward deflection designates reduction.
400 nmol of ADP resulted in the transient oxidation of the cytochrome (state III) followed by reduction back to the pre-ADP level (state IV). Alternating the concentration of ADP had no effect on the resulting reduction state but proportionally altered the time spent at the state III redox state. The cytochromes were then reduced by the addition of KCN. At all wavelength pairs and in both euthyroid and hyperthyroid mitochondria, the percent reduction of each cytochrome during states III and IV was mitochondrial protein-independent while the changes in absorption were directly proportional to protein concentration (data not shown). Hyperthyroidism resulted in large increases in TABLE
2
CYTOCHROME CONCENTRATIONS CHONDRIA ISOLATED FROM THE EUTHYROID OR HYPERTHYROID RATS Cytochrome
b Cl
c Total c a 03
the concentration of some, but not all, of the mitochondrial cytochromes. Table 2 shows that cytochromes 6, c, total c and a a3 increased in content in mitochondria isolated from hyperthyroid rats. The concentration of cytochrome c, in the hyperthyroid rat mitochondria was not significantly different from that of euthyroid rat mitochondria. A comparison of the concentrations of total cytochrome c and the sum of cytochromes ci and c found no significant difference between them (data not shown). Thus, the increase in concentration of total cytochrome c is due to an increase in the concentration of cytochrome c and not of cytochrome c,. The determination of cytochrome concentration by the above manner has several possible problems. The major potential errors are the overlaps in the spectra of cytochromes c and c, and the use of succinate and KCN to reduce the cytochromes. To resolve these difficulties, mitochondria were isolated and underwent exhaustive extraction of cytochrome c by 5 cycles of freeze-thawing the mitochondria in 0.9% KCl. Extraction of cytochrome c from these membranes was completed by the 5th cycle (data not shown). Difference spectra, using sodium dithionite as the reducing agent, were then performed on the cytochrome c-depleted membranes. Table 3 shows that cytochromes b and a a3 increased in concentration in hyperthyroid rats while the concentration of cytochrome c, remained constant. These data demon-
Concentration (nmol cyt/mg
IN MITOLIVERS OF
mito protein)
Euthyroid (n =lO)
Hyperthyroid (n=8)
0.09f0.01 0.17 f 0.02 0.18f0.01 0.32 f 0.02 0.29f0.03
0.16 fO.O1 0.18 f 0.02 0.29 f 0.03 0.46*0.03 0.54 f 0.04
Values represent the mean f standard NS = not significantly different. * =P
error.
TABLE
3
EFFECTS OF THYROXINE SPECIFIC CYTOCHROMES PLETED MITOCHONDRIAL Cytochrome
* NS * * *
b Cl
a
a3
Cytochrome
ON THE CONTENT OF IN CYTOCHROME c-DEMEMBRANES content
a
Euthyroid (n=9)
Hyperthyroid (n=7)
0.81 f 0.20 1.67kO.11 1.45 *0.12
1.56kO.20 1.79k0.17 2.52k0.13
* NS *
Values represent the mean f standard error. NS = Not significantly different. * =P
pairs
161 TABLE
4
THYROXINE-INDUCED CHANGES IN THE PERCENT REDUCTION OF THE MITOCHONDRIAL CYTOCHROMES DURING STATE III AND IV RESPIRATION WITH SUCCINATE Cytochromes
b cl c Total c a3
Percent reduction State IV
State III Euthyroid (n=13)
Hyperthyroid (n=17)
Euthyroid (n =13)
Hyperthyroid (n =17)
19.0f0.9 19.1k1.9 19.3kl.l
23.4i-0.9 24.8k0.9 25.7kO.8
* * *
19.1f1.2 13.0f0.9
26.2f0.8 14.71t0.9
* NS
61.7kO.9 30.2kO.7 28.3kl.l 30.7f0.8 22.9jcO.7
49.5k0.7 37.9k0.6 38.9f0.6 40.9rtO.7 23.9f0.7
Values represent the meanf standard NS = Not significantly different. * =P<0.001. n = Number of animals per group.
* * * * NS
error.
strate that the mitochondrial cytochrome chain does not respond as a structural unit in the hyperthyroid state. Mitochondria isolated from hyperthyroid rats not only showed changes in oxygen consumption (Table 1) and in cytochrome content (Tables 2 and 3), but also in the percent reduction of the mitochondrial cytochromes. Table 4 shows the percent reduction of the cytochromes during state III and IV respiration. During state III respiration cytochromes b, cl, c and total c were more reduced by succinate in the hyperthyroid mitochondria, while there was no significant change in the percent reduction of cytochrome a a3. During state IV respiration cytochromes c,, c and total c were also more reduced in hyperthyroid mitochondria. But, in contrast to the increase seen in state III respiration, the percent reduction of cytochrome b decreased during state IV respiration. Thus, hyperthyroidism not only results in changes in the concentration of the cytochromes but also in their reduction state. Discussion The increase in respiration observed in mitochondria isolated from hyperthyroid rats is well known (Drabkin, 1949; Lardy and Feldott, 1951; Martius and Hess, 1951). Thyroid status not
only regulates respiration, but also controls the concentration of electron transport components, such as ubiquinone (Edwin et al., 1960), the cytochromes (Drabkin, 1949; Edwin et al., 1960; Roodyn et al., 1965; Booth and Holloszy, 1975; Winder and Holloszy, 1977; Nishiki et al., 1978) and associated enzymes such as the atractylosidesensitive adenine nucleotide carrier (Babior et al., 1973; Portnay et al., 1973). Information of this sort led Nishiki et al. (1978) to propose that the components of the respiratory chain respond as a respiratory unit to the animals’ thyroid status. However, increased respiration can also occur in the absence of changes in cytochrome content but in the presence of more reduced cytochromes (Bronk, 1966). In order to determine the possible roles of these processes on the hormone-induced increased respiration, we have determined the concentration of each cytochrome and it’s redox state during state III and IV respiration. Hypothyroid rats have decreased concentration of cytochromes b, c and a a3 which increase with thyroid hormone replacement, lowered respiratory rates, and less reduced cytochrome states (Drabkin, 1949; Edwin et al., 1960; Roodyn et al., 1965; Bronk, 1966; Nishiki et al., 1978; Sterling et al., 1980). Hyperthyroid rats have elevated concentrations of cytochromes b, c and a a3 (Table 2; Drabkin, 1949; Edwin et al., 1960; Booth and Holloszy, 1975; Winder and Holloszy, 1977; Nishiki et al., 1978). These data would tend to support the regulation of cytochrome concentration by thyroid hormones through a unit response. But, in these studies, cytochrome c was measured either as total cytochrome c + c, at 550-540 nm (Drabkin, 1949; Edwin et al., 1960; Roodyn et al., 1965; Bronk, 1966; Nishiki et al., 1978) or as isolated cytochrome c (Booth and Holloszy, 1975; Winder and Holloszy, 1977). We find (Tables 2 and 3) that the concentration of cytochrome c, is unaltered by T., treatment and that the increase in cytochrome c + c, is due to an increase in the concentration of cytochrome c and not of cytochrome c,. This differential response of ci has also recently been demonstrated in mitochondria isolated from rat hepatocyte cultures treated with triiodothyronine (Tj) (Wilson and McMurray, 1983). They found elevated concentrations of cytochrome c, b and a a3 while the concentration of
168
ci remained unchanged. Thus, it appears that the regulation of cytochrome c, concentration differs from the regulation of the other cytochromes in hyperthyroidism. The increased oxidative capabilities of mitochondria isolated from hyperthyroid rats cannot entirely be due to increased respiratory units, as has been suggested (Nishiki et al., 1978) since cytochrome c, concentration remained constant. The rate of respiration is also related to the reduction state of the cytochromes. Within 3 h after T3 treatment of hypothyroid rats, mitochondrial respiration increased, cytochrome concentrations remained constant, and cytochromes b, c + cl and a a3 became more reduced (Bronk, 1966). 48 h after T3 treatment, respiration was further elevated (Tata et al., 1963; Bronk, 1966), cytochrome concentration remained virtually unchanged (Roodyn et al., 1965; Bronk, 1966) and all of the cytochromes, except b, continued to become more reduced (Bronk, 1966). These data led Bronk (1966) to propose that these increases in oxidative activity were not due to changes in the concentration of electron transport components but rather appeared due to an effect of Ts on the rate of reduction of the cytochromes. The relationship between increased respiration and an increased redox state of the cytochromes has also recently been observed for glucagontreated rats (Halestrap, 1982). Mitochondria isolated from the livers of glucagon-treated rats showed enhanced succinate respiration. Cytochrome spectra performed on these mitochondria demonstrated increased reduction of cytochromes b, cl and c during state III and state IV respiration. This enhanced reduction appears due to increased substrate availability for mitochondrial respiration (Kimura et al., 1984). How the hyperthyroid electron transport chain responds to the increased rate of cytochrome reduction apparently depends on the metabolic state of the mitochondria. We found in state III respiration, when respiration is limited by substrate oxidation (Chance, 1965) that cytochromes b, cl, c and c + ci were more reduced. This increased redox state appears due to enhanced electron flow from the site of substrate oxidation. A major controlling step for electron transport is at the site of pyridine nucleotide oxidation (Chance, 1965). In
hyperthyroid rat hearts, the mitochondrial [NAD]/[NADH] was elevated compared to control hearts, suggesting augmented electron flow from NADH (Nishiki et al., 1978). In our experiments, electrons from NADH were blocked by rotenone but the increased activity of succinate dehydrogenase indicates increased electron flow from this substrate. Succinate dehydrogenase activity increased in the T,-treated animals by approximately 27% while cytochromes b, cl and c were approximately 27% more reduced. Our data indicates that an equilibrium exists between the site of substrate oxidation and the redox states of cytochromes b, c and c, during state III in the hyperthyroids. During state IV respiration, when respiration is controlled by ADP availability (Chance, 1965) the relative oxidation of cytochrome b indicates the electron transport chain is in disequilibrium in the T,-treated animals. The apparent cause of this disequilibrium may be ‘loose coupling’ as suggested by Hassinen et al. (1971). These investigators found evidence for ‘loose coupling’ of the electron transport chain near cytochrome b in intact livers from hyperthyroid rats, wherein there was no inhibition of respiration by fructose and there was no cross-over point between the flavoproteins, indicators of the redox state of the nicotinamide nucleotides, and cytochrome b. Our data is also consistent with this concept of loose coupling. If loose coupling occurs it should be most apparent during state IV. We find that cytochrome b is more oxidized in state IV, as would be expected in ‘loose coupling’. We also find the relative changes in the rates of rnitochondrial respiration are greater during state IV respiration (Table 1). The rate of state IV respiration in the hyperthyroids was nearly 40% greater than the euthyroid rate, while the rate of state III respiration was increased by approximately 20% in the hyperthyroid mitochondria. These 2 observations support the concept that T4 treatment results in ‘loose coupling’ and this alteration occurs at or near cytochrome b. The disequilibrium of the electron transport chain during state IV respiration in the hyperthyroids is also seen in the more reduced redox state of cytochrome ci in contrast to the relative oxidation of b. The mechanism responsible for this
169
equilibrium remains unclear but may involve the transfer of electrons from an increased content of cytochrome b to an unchanged content of c,. The result of these T4-induced changes in the concentration and the redox states of the cytochromes is to increase respiration capacity. T4 treatment increases respiration through several mechanisms. One mechanism may be via loose coupling of the chain near cytochrome b. This would increase respiration when substrates were plentiful but ADP was not. Another mechanism is increasing the activity of cytochrome oxidase. We find that T4 induces an increased concentration of cytochrome a u3, components of cytochrome oxidase (Tables 2 and 3) and a more reduced redox state for cytochrome c, a rate-listing step for cytochrome oxidase (Nishiki et al., 1978). These thyroxine-induced changes could result in increased oxidase activity and increased respiration, especially when neither substrate nor ADP is limiting. Direct in vivo evidence for increased ATP synthesis is lacking. However, Tata et al. (1963) have shown that physiological doses of T4 produce enhanced metabolic rates and ~tochond~al respiratory rates without uncoupling of oxidative phosphorylation. Loose coupling and enhanced respiratory capability are potential mechanisms for thyrotoxic mitochondria to meet the increased energy demands of the whole animal. References Ackrell, B.A.C., Keamey, E.B. and Singer, T.P. (1978) Methods Enzymol. 53, 446-482. Babior, B.M., Creagan, S., Ingbar, S.H. and Kipnes, R.S. (1973) Proc. Natl. Acad. Sci. (U.S.A.) 70, 98-102. Booth, F.W. and Holloszy, J.O. (1975) Arch. Biochem. Biophys. 167, 674-677. Bradford, M. (1976) Analyt. Biochem. 72, 248-254. Bronk, J.R. (1966) Science 153. 638-639. Chance, B. (1965) In: Control of Energy Metabolism, Eds.: B.
Chance, R.W. Estabrook and J.R. WiIli~~n (Academic Press, New York) pp. 415-435. Chance, B. and Williams, G.R. (1956) In: Advances in Enzymology, Vol. 17, Ed: F.F. Nord (Interscience Publishers, New York) pp. 65-134. Drabkin, D.L. (1949) J. Biol. Chem. 182, 335-349. Edwin, E.E., Green, J., Diplock, A.T. and Bunyan, J. (1960) Nature (Lond.) 186, 725. Erecinska, M. and Wilson, D.F. (1982) J. Membr. Biol. 70, l-14. Halestrap, A.P. (1982) Biochem. J. 204, 37-47. Hassinen, I.E., Ylikahri, R.H. and Kahonen, M.T. (1971) Arch. Biochem. Biophys. 147, 255-261. Horrum, M.A. and Tobin, R.B. (1983) Fed. Proc. 42, 325. Johnson, D. and Lardy, H.A. (1967) Methods Enzymol. 10, 94-95. Kimura, S., Suzaki, T., Kobyashi, S., Abe, K. and Ogata, E. (1984) Biochem. Biophys. Res. Commun. 119, 212-219. Lardy, H.A. and Feldott, G. (1951) Ann, N.Y. Acad. Sci. 54, 531-536. Martius, C. and Hess, B. (1951) Arch. Biochem. Biophys. 33, 486-487. Nishiki, K., Erecinska, M., Wilson, D.F. and Cooper, S. (1978) Am. J. Physiol. 235, &212-C219. Portnay, G.I., McClendon, F.D., Bush, J.E., Braverman, L.E. and Babior, B.M. (1973) Biochem. Biophys. Res. Commun. 55,17-20. Roodyn, D.B., Freeman, K.B. and Tata, J.R. (1965) Biochem. J. 94, 628-641. Siess, E.A. (1983) Hoppe-Seylers Z. Physiol. Chem. 364, 279-290. Sterling, K., Brenner, M.A. and Sakurda. T. (1980) Science 210. 340-342. Tata, J.R., Emster, L., Lindberg, O., Arrhenius, E., Pedersen, S. and Hedman, R. (1963) Biochem. J. 86, 408-428. Tobin, R.B., Mackerer, CR. and Mehlman, M.A. (1972) Am. J. Physiol. 223, 83-88. Vanneste, W.H. (1965) Biochim. Biophys. Acta 113, 175-178. Wemette, M.E., Ochs, R.S. and Lardy, H.A. (1981) J. Biol. Chem. 256, 12767-12771. Williams, J.N. (1964) Arch. Biochem. Biophys. 107, 537-543. Wilson, E.J. and McMurray, W.C. (1983) Can. J. Biochem. Cell Biol. 61, 636-643. Wilson, D.F., Owen, C.S. and Holian, A. (1977) Arch. Biothem. Biophys. 167,749-762. Winder, W.W. and HoIIoszy, J.O. (1977) Am. J. Physid. 232, C18OX184.