Biochemical and stereological analysis of rat liver mitochondria in different thyroid states

The concentrations of the inner mitochondrial membrane markers cardiolipin and cytochrome alpha have been measured in liver homogenates and in purified mitochondria after thyroxine administration to thyroidectomized and normal rats. The biochemical results have been correlated with stereological electron micrographic analyses of hepatocytes in liver sections, and of isolated mitochondrial pellets. There were progressive and parallel increases in homogenate and mitochondrial cardiolipin concentration, and in mitochondrial cytochrome alpha concentration, after administration of 20 microgram of thyroxine on alternate days to thyroidectomized rats, and of 300 microgram on alternate days to normal rats. Electron microscope measurements showed marked differences in the shape of the mitochondria and in the number of cristae in different thyroid states. Hypothyroid mitochondria were shorter and wider than controls, and hyperthyroid mitochondria longer but of similar width. Mitochondrial volume per unit cell volume was virtually unchanged in hypo- and hyperthyroid animals. The most striking changes were a decrease in the area of the inner membrane plus cristae in thyroidectomized rats, and a substantial increase in membrane area after thyroxine administration. The biochemical and electron micrographic results indicate that, in rat liver, thyroid hormone administration leads to a selective increase in the relative amount of mitochondrial inner membranes, with little or no change in the mitochondrial volume per unit cell volume, or in total mitochondrial protein per unit total cell protein.     

Effects of L-malate on mitochondrial oxidoreductases in liver of aged rats

Accumulation of oxidative damage has been implicated to be a major causative factor in the decline in physiological functions that occur during the aging process. The mitochondrial respiratory chain is a powerful source of reactive oxygen species (ROS), considered as the pathogenic agent of many diseases and aging. L-malate, a tricarboxylic acid cycle intermediate, plays an important role in transporting NADH from cytosol to mitochondria for energy production. Previous studies in our laboratory reported L-malate as a free radical scavenger in aged rats. In the present study we focused on the effect of L-malate on the activities of electron transport chain in young and aged rats. We found that mitochondrial membrane potential (MMP) and the activities of succinate dehydrogenase, NADH-cytochrome c oxidoreductase and cytochrome c oxidase in liver of aged rats were significantly decreased when compared to young control rats. Supplementation of L-malate to aged rats for 30 days slightly increased MMP and improved the activities of NADH-dehydrogenase, NADH-cytochrome c oxidoreductase and cytochrome c oxidase in liver of aged rats when compared with aged control rats. In young rats, L-malate administration increased only the activity of NADH-dehydrogenase. Our result suggested that L-malate could improve the activities of electron transport chain enzymes in aged rats.     

Mitochondrial respiration and triiodothyronine concentration in liver from postpubertal and adult rats.

The purpose of this study was to investigate the decline in rat liver mitochondria respiration found in adult rats compared to younger ones, and to find a link between this respiratory impairment and a tissue hypothyroidism state. To this end, hepatic concentration and serum levels of triiodothyronine were measured in postpubertal rats (60 days old) and adult rats (180 days old). In addition, in these rats we measured oxidative phosphorylation in homogenate together with coupled and uncoupled respiration in isolated mitochondria using succinate or durohydroquinone as substrate. We found that mitochondria from adult rats consumed less oxygen compared to younger rats due to lower electron transport chain and phosphorylating system activity. In addition, we found that in state 4 condition, mitochondria from adult rats consumed less oxygen than mitochondria from young rats. Finally, we found a decrease in liver triiodothyronine concentration in adult rats. In conclusion, the results of this study show that hepatic mitochondria in adult rats have a decreased ATP synthesis capacity and proton permeability, both consistent with the tissue hypothyroidism found in the liver of adult rats.     

Studies on the effects of copper deficiency on rat liver mitochondria. I. Changes in mitochondrial composition

As part of an investigation of the lesions of copper (Cu) deficiency a study was undertaken of the copper, iron, cytochrome and fatty acid composition of liver mitochondria from Cu deficient and Cu-adequate control rats. Cu concentrations were significantly decreased in whole liver, liver mitochondria and in blood plasma. Total iron was significantly increased in whole liver but remained at the normal level in mitochondria. Cytochrome c oxidase (EC 1.9.3.1) and its component cytochromes a and a3 were significantly reduced in liver mitochondria from Cu-deficient rats, whereas there was no effect on the concentration of cytochromes b, c1 and c. Evidence from comparisons between cytochrome c oxidase activity and the amount of enzyme present, as assessed from the mitochondrial cytochrome a and a3 content, suggests that in addition to an absolute loss of enzyme, Cu-deficiency adversely affects the efficiency of the residual enzyme. Severe Cu deficiency had no effect on 'ageing' or 'swelling' properties of liver mitochondria, indicating no marked effects on fatty acid composition. Fatty acid analyses demonstrated a slight but significant increase in docosapentenoic acid (22:5) of Cu-deficient mitochondria, but since this represents a minor component there was no change observed in the 'unsaturation index'. It was concluded that, in contrast to previous reports, Cu deficiency of the severity reported did not have a deleterious effect on the integrity and permeability of the inner mitochondrial membrane as exemplified by any qualitative modification of fatty acid constitution per se.     

Decreased activities of ubiquinol:ferricytochrome c oxidoreductase (complex III) and ferrocytochrome c:oxygen oxidoreductase (complex IV) in liver mitochondria from rats with hydroxycobalamin[c-lactam]-induced methylmalonic aciduria.

Rats treated with hydroxycobalamin[c-lactam] (HCCL), a cobalamin analogue that induces methylmalonic aciduria, have increased hepatic mitochondrial content and increased oxidative metabolism of pyruvate and palmitate per hepatocyte. The present studies were undertaken to characterize oxidative metabolism in isolated liver mitochondria from rats treated with HCCL. After 5-6 weeks, state 3 oxidation rates for diverse substrates are reduced in mitochondria from HCCL-treated rats. Similar reductions of mitochondrial oxidation rates are obtained with dinitrophenol-uncoupled mitochondria excluding defective phosphorylation as a cause for the observed decrease in mitochondrial oxidation. The activities of mitochondrial oxidases are reduced in HCCL-treated rats and demonstrate a defect in complex IV. Investigation of the complexes of the respiratory chain reveals a 32% decrease of ubiquinol:ferricytochrome c oxidoreductase (complex III) activity and a 72% decrease of ferrocytochrome c:oxygen oxidoreductase (complex IV) activity in mitochondria from 5-6-week HCCL-treated rats as compared with controls. Liver mitochondria from HCCL-treated rats also demonstrate decreased cytochrome content per mg of mitochondrial protein (25% decrease of cytochrome b and 52% decrease of cytochrome a + a3 as compared with control rats). The HCCL-treated rat represents an animal model for the study of the consequences of respiratory chain defects in liver mitochondria.     

Influence of starvation and clofibrate administration on oxidative phosphorylation by rat liver mitochondria.

Whole cells, homogenates and mitochondrial obtained from the livers of albino rats which were starved for 6 days or more showed a 50% decrease in oxidative activity. The decrease could be corrected by the addition of cytochrome c in vitro. The phosphorylative activity of mitochondria remained unaffected. The decrease in oxidative rate was not observed when starving animals were given the anti-hypercholesterolaemic drug clofibrate. The total cellular concentration of cytochrome c was not affected by starvation. However, the concentration of the pigment in hepatic mitochondria isolated from starving animals was less than half that in normal mitochondria. Clofibrate-treated animals did not show a decreased concentration of cytochrome c in hepatic mitochondria. Mitochondria isolated from starving animals, though deficient in cytochrome c, did not show any decrease in succinate dehydrogenase activity or in the rate of substrate-dependent reduction of potassium ferricyanide or attendant phosphorylation. In coupled mitochondria, ferricyanide may not accept electrons from the cytochrome c in the respiratory chain. Starvation decreases the concentration of high-affinity binding sites for cytochrome c on the mitochondrial membrane. The dissociation constant increases in magnitude.     

Mitochondrial development in liver of foetal and newborn rats

THE DEVELOPMENT OF THE INNER MITOCHONDRIAL MEMBRANE IN FOETAL AND NEONATAL RAT LIVER WAS STUDIED BY FOLLOWING THREE PARAMETERS: (1) the activity of several respiratory enzymes in homogenates and purified mitochondria, (2) the spectrophotometric determination of cytochrome content in the mitochondria and (3) the cardiolipin content in both homogenates and purified mitochondria. Respiratory-enzyme activities of homogenates of foetal liver were one-quarter to one-twentieth of those of homogenates of adult liver, and the enzyme specific activities in purified mitochondria from foetal liver were one-half to one-eighth of those in mitochondria from adult liver. The cardiolipin content of liver homogenates increased approximately twofold during the development period, but there was no significant change in the cardiolipin content of purified mitochondria. It is concluded that cell mitochondrial content approximately doubles in the immediate postnatal period. There was no evidence for an increase in the relative amount of cristae protein in mitochondria during this period to account for increases in mitochondrial enzyme specific activity, since cardiolipin and cytochrome concentrations remained unchanged and electron micrographs revealed no differences. The cause of the lower respiratory-enzyme specific activity in foetal liver mitochondria is unclear. Qualitative differences in respiratory units in foetal and mature animals are suggested.     

Interrelationships of copper, cytochrome oxidase, phospholipid synthesis and adenine nucleotide binding.

Studies are reported on the interrelationships in liver mitochondria of copper status, cytochrome oxidase activity, adenine nucleotide binding capacity and phospholipid synthesis. Direct exposure of mitochondria to cyanide or diethyldithiocarbamate depressed cytochrome oxidase activity; ADP-binding and phospholipid synthesis. Fractionation of mitochondria to increase the specific activity of cytochrome oxidase about 10-fold did not increase the affinity to bind ADP. Ageing of mitochondria or dialysis of mitochondria or mitochondrial membrane preparations against water or diethyldithiocarbamate at 0--2 degrees for 18 h did not decrease cytochrome oxidase activity or copper content of reisolated and resuspended mitochondria or mitochondrial membrane preparations, but considerably reduced the affinity to bind ADP. The respiratory inhibitors, fluoride and azide, at concentrations inhibitory to cytochrome oxidase did not reduce ADP-binding or phospholipid synthesis. Atractyloside did not inhibit cytochrome oxidase activity but did inhibit ADP-binding and phospholipid synthesis. Pre-incubation of mitochondrial membrane preparations with Cu++ increased the copper content and ADP-binding affinity. The results indicate that cytochrome oxidase is not the ADP-binding site of the mitochondrial membrane system and that reduced cytochrome oxidase activity per se does not depress binding affinity. Copper appears to be a component of the adenine nucleotide binding sites of mitochondrial membranes because the copper-complexing agents, cyanide and diethyldithiocarbamate, depressed ADP-binding, while increased mitochondrial membrane copper content increased ADP-binding.     

Aging-related decreases in hepatic mitochondrial and cytosolic δ-aminolevulinic acid synthase during experimental porphyria

The basal- and allylisopropylacetamide-induced activities of the first enzyme of heme biosynthesis, δ-aminolevulinic acid synthase (ALAS) were measured in hepatic mitochondria and cytosol of young, adult, and aged Fisher 344 rats. The total cellular ALAS activity induced by allylisopropylacetamide decreased 67% with age. The specific activity of mitochondrial ALAS in normal and induced animals decreased with aging when assayed in whole or broken mitochondria. The levels of ALAS which accumulated in the cytosol after allylisopropylacetamide administration were proportionally greater in both the young and senescent than in the mature animals. During aging, no evidence for a fragile population of mitochondria in either normal or induced animals was observed suggesting that mitochondrial matrix proteins are not released during homogenization. The hepatic mitochondrial content decreased during aging when calculated using both a membrane-bound marker enzyme cytochrome oxidase and a matrix marker enzyme citrate synthase and was unaffected by allylisopropylacetamide treatment. This reduced mitochondrial content further diminishes the level of functional ALAS available in the liver during senescence. This study confirms the age-dependent decrease in mitochondria ALAS in normal and induced animals and also suggests an age-related change in the process by which cytosolic ALAS is translocated into the mitochondria.     

Protamine inhibition of the oxidative phosphorylation in intact, cytochrome c-depleted and restored mitochondria.

On the basis of polarographic data it is shown that protamine has a biphasic effect on the respiration of intact mitochondria. At lower protamine concentrations respiration is stimulated and this combined with a decrease of the respiratory control index; at higher ones respiration is inhibited and respiratory control is lost. In cytochrome c-depleted and restored mitochondria protamine effect on oxidative phosphorylation is only inhibitory. Increasing cytochrome c concentrations restore respiration in protamine-treated cytochrome c depleted mitochondria but not the respiratory control. Binding of cytochrome c to mitochondria is studied by determining from Scatchard plots the number of high affinity binding sites (n) and their stability constants (K). In absence of protamine in intact mitochondria n = 2.7 and K = 4.67-10(6) M-1; in cotochrome c depleted mitochondria n = 4.7 and K = 5.16-10(6) M-1. In both types of mitochondria protamine decreases significantly n as well as K. These data show that protamine may affect oxidative phosphorylation by causing desorption of cytochrome c from the inner mitochondrial membrane.     

Toxicity of Copper on Isolated Liver Mitochondria: Impairment at Complexes I,II, and IV Leads to Increased ROS Production

Oxidative damage has been implicated in disorders associated with abnormal copper metabolism and also Cu²⁺ overloading states. Besides, mitochondria are one of the most important targets for Cu²⁺, an essential redox transition metal, induced hepatotoxicity. In this study, we aimed to investigate the mitochondrial toxicity mechanisms on isolated rat liver mitochondria. Rat liver mitochondria in both in vivo and in vitro experiments were obtained by differential ultracentrifugation and the isolated liver mitochondria were then incubated with different concentrations of Cu²⁺. Our results showed that Cu²⁺ induced a concentration and time-dependent rise in mitochondrial ROS formation, lipid peroxidation, and mitochondrial membrane potential collapse before mitochondrial swelling ensued. Increased disturbance in oxidative phosphorylation was also shown by decreased ATP concentration and decreased ATP/ADP ratio in Cu²⁺-treated isolated mitochondria. In addition, collapse of mitochondrial membrane potential (MMP), mitochondrial swelling, and release of cytochrome c following of Cu²⁺ treatment were well inhibited by pretreatment of mitochondria with CsA and BHT. Our results showed that Cu²⁺ could interact with respiratory complexes (I, II, and IV). This suggests that Cu²⁺-induced liver toxicity is the result of metal’s disruptive effect on liver hepatocyte mitochondrial respiratory chain that is the obvious cause of Cu²⁺-induced ROS formation, lipid peroxidation, mitochondrial membrane potential decline, and cytochrome c expulsion which start cell death signaling.     

The oxygen dependence of mitochondrial oxidative phosphorylation measured by a new optical method for measuring oxygen concentration

Oxygen-dependent quenching of phosphorescence has been used to measure the dependence of mitochondrial oxidative phosphorylation on oxygen concentration in suspensions of isolated rat liver mitochondria. An instrument has been designed which simultaneously monitors the phosphorescence lifetime of a fluorophor and the reduction of cytochrome c by dual wavelength spectrophotometry. The phosphorescence lifetime method gives very rapid (less than 100 ms) measure of the oxygen concentration (Vanderkooi, J. M., Maniara, G., Green, T. J., and Wilson, D. F. (1987) J. Biol. Chem. 262, 5476-5482) from concentrations characteristic of air-saturated media to as low as 2 x 10(-8) M. The results may be summarized as follows. For well coupled rat liver mitochondria at pH 7.0 and in the presence of ATP, as the oxygen concentration was lowered, increased cytochrome c reduction was observed to begin at oxygen concentrations greater than 20 microM. For mitochondria in the presence of uncoupler, cytochrome c reduction began at oxygen concentrations less than 1.0 microM. The oxygen dependence of reduction of cytochrome c in well coupled mitochondria treated with ATP was strongly dependent on the pH of the suspending medium. Reduction of cytochrome c began at higher oxygen concentrations as the pH was made more alkaline. The oxygen concentration for half-maximal respiratory rates was much larger for well coupled mitochondria treated with ATP (approximately 0.7 microM) than for mitochondria treated with uncoupler (less than 0.1 microM). It is concluded that the oxygen dependence of mitochondrial oxidative phosphorylation is such that mitochondria could function in their proposed role of tissue oxygen sensors for regulation of such diverse functions as local blood flow and electrical activity in the carotid body.     

Liver lipase and high-density lipoprotein. Lipoprotein changes after incubation of human serum with rat liver lipase

Under standard conditions, liver regeneration is impaired if mitochondrial protein synthesis is completely blocked. By treating rats with oxytetracycline for various periods of time directly prior to partial hepatectomy, livers were led to a condition of relative deficiency in cytochrome c oxidase and ATP synthetase. To this end, oxytetracycline was administered by means of continuous intravenous infusion up to concentrations of 20 micrograms/ml serum, giving a gradual decrease in cytochrome c oxidase activity. This activity was used as a marker for functionally capable mitochondria and as a tool to monitor the efficiency of inhibition of mitochondrial protein synthesis. It is shown that liver regeneration is strongly impaired after a period of pretreatment of 22 days or more and continuation of oxytetracycline treatment during regeneration. The mitochondrial respiratory capacity is reduced to 14% of the control value under these conditions. To obtain inhibitory levels within the regenerating liver, it was necessary to raise the serum levels slightly above 20 micrograms/ml. This measure is most likely required because of the poor vascularization of the regenerating liver. The serum levels were kept, however, far below those known to inhibit cytoplasmic protein synthesis. The results show that in normal liver the respiratory capacity must be reduced drastically before energy-requiring processes become affected. In Zajdela hepatoma cells, similar effects are found after reduction of the cytochrome c oxidase activity to 38%. This difference in sensitivity is probably based on the different mitochondrial content of liver cells and the liver-derived Zajdela cells.     

Functions of cytochrome c in regulation of electron transfer and protein folding.

Cytochrome c, a "mobile electron carrier" of the mitochondrial respiratory chain, also occurs in detectable amounts in the cytosol, and can receive electrons from cytochromes present in endoplasmic reticulum and plasma membranes as well as from superoxide and ascorbate. The pigment was found to dissociate from mitochondrial membranes in liver and kidney when rats were subjected to heat exposure and starvation, respectively. Treating cytochrome c with hydroxylamine gives a partially deaminated product with altered redox properties; decreased stimulation of respiration by deficient mitochondria, increased reduction by superoxide, and complete loss of reducibility by plasma membranes. Mitochondria isolated from brown adipose tissue of cold-exposed rats are found to be sub-saturated with cytochrome c. The ability of cytochrome c to reactivate reduced ribonuclease is now reinterpreted as a molecular chaperone role for the hemoprotein.     

The effect of sodium selenite toxicity on tissue distribution of zinc,iron, and copper in rats

Male Sprague-Dawley rats were used to determine the effects of suptoxic and toxic concentrations of selenite in the drinking water on tissue distribution of zinc (Zn), iron (Fe), and copper (Cu). Se (as sodium selenite) was provided in drinking water at concentrations of 0, 2, 4, and 8 ppm. At 19 d, half of the rats in 4 and 8 ppm Sesupplemented groups were kept on drinking water alone for additional 13 d. All rats were sacrificed at the end of 32 d of experiment. Heart, liver, and kidney were analyzed for the concentrations of Fe, Zn, and Cu by atomic absorption spectrophotometry and of Se by a fluorometric method. Results indicated that rats receiving 4 and 8 ppm Se in drinking water showed a marked reduction in food intake and a reduced growth rate. These adverse effects were quickly reversed when high Se intake was discontinued. Se toxicity caused minimal change in zinc status, reduced tissue iron concentrations and caused a marked increase in copper contents in heart, liver, and kidney. The latter findings were only partly reversed after removal of Se in drinking water. The accumulation of Cu in the tissues of Se-toxic rats provides the evidence of some interaction between Se and Cu.     

Improving effects obtained by the ovariectomy or treatment with tamoxifen of female diabetic rats over the function and enzyme activities of liver mitochondria.

In the present work we studied, in female chronic diabetic rats the effect of either the parenteral administration of tamoxifen (TAM) (500 micrograms.kg-1.day-1) for 15 days or the ovariectomy upon the respiration and oscillatory behaviour of intact mitochondria and the activities of 3-hydroxybutyrate dehydrogenase (HBD) and cytochrome c oxidase (Cox) of disrupted liver mitochondria. The treatment with TAM as well as the ovariectomy of diabetic animals significantly increased the respiratory control (RC) and the state 3 (S3) of respiration of intact liver mitochondria with the three substrates assayed (3-hydroxybutyrate, malate-glutamate and succinate). Both treatments also lowered significantly the damped factors of the oscillatory variation of liver intact mitochondria of diabetic rats. Moreover, the two above-mentioned treatments restored the activities of HBD and Cox of liver disrupted mitochondria to normal values. The effect of estrogens at level of its receptors in the modulation of liver mitochondrial function and liver HBD and Cox activities in chronic diabetes is discussed.     

Stimulation of the electron transport chain in mitochondria isolated from rats treated with mannoheptulose or glucagon

We investigated the kinetics of the mitochondrial respiratory chain, proton leak, and phosphorylating subsystems of liver mitochondria from mannoheptulose-treated and control rats. Mannoheptulose treatment raises glucagon and lowers insulin; it had no effect on the kinetics of the mitochondrial proton leak or phosphorylating subsystems, but the respiratory chain from succinate to oxygen was stimulated. Previous attempts to detect any stimulation of cytochrome c oxidase by glucagon are shown by flux control analysis to have used inappropriate assay conditions. To investigate the site of stimulation of the respiratory chain we measured the relationship between the thermodynamic driving force and respiration rate for the span succinate to coenzyme Q, the cytochrome bc1 complex and cytochrome c oxidase. Hormone treatment of rats altered the kinetics of electron transport from succinate to coenzyme Q in subsequently isolated mitochondria and activated succinate dehydrogenase. The kinetics of electron transport through the cytochrome bc1 complex were not affected. Effects on cytochrome c oxidase were small or nonexistent.     

The maturation of the inner membrane of foetal rat liver mitochondria.

A new method was devised for the isolation of foetal and neonatal rat lvier mitochondria, giving higher yields than conventional methods. 2. During development from the perinatal period to the mature adult, the ratio of cytochrome oxidase/succinate-cytochrome c reductase changes. 3. The inner mitochondrial membrane of foetal liver mitochondria possesses virtually no osmotic activity; the permeability to sucrose decreases with increasing developmental age. 4. Foetal rat liver mitochondria possess only marginal respiratory control and do not maintain Ca2+-induced respiration; they also swell in respiratory-control medium in the absence of substrate. ATP enhances respiratory control and prevents swelling, adenylyl imidodiphosphate, ATP+atractyloside enhance the R.C.I. (respiratory control index), Ca2+-induced respiratory control and prevent swelling, whereas GTP and low concentrations of ADP have none of these actions. It is concluded that the effect of ATP depends on steric interaction with the inner mitochondrial membrane. 5. When 1-day pre-partum foetuses are obtained by Caesarean section and maintained in a Humidicrib for 90 min, mitochondrial maturation is "triggered", so that their R.C.I. is enhanced and no ATP is required to support Ca2+-dependent respiratory control or to inhibit mitochondrial swelling. 6. It is concluded that foetal rat liver mitochondria in utero do not respire, although they are capable of oxidative phosphorylation in spite of their low R.C.I. The different environmental conditions which the neonatal rat encounters ex utero enable the hepatic mitochondria to produce ATP, which interacts with the inner mitochondrial membrane to enhance oxidative phosphorylation by an autocatalytic mechanism.     

Time-course of effects of toluene on microsomal enzymes in rat liver, kidney and lung during and after inhalation exposure

Inhalation of toluene vapour of 2000 ppm increased the activities of aniline hydroxylase, aminopyrine N-demethylase, aryl hydrocarbon hydroxylase and NADPH-cytochrome c reductase and the concentrations of cytochromes P-450 and b5 in liver microsomes of adult male rats after an exposure period of 1 day or less. Repeated treatments, 8 h daily for 1-16 days, had only a slight further effect. In lung microsomes, the activities of monooxygenases and the concentration of cytochrome P-450 decreased after 6-24 h toluene exposure, but those of cytochrome b5 and NADPH-cytochrome c reductase did not change. In kidney microsomes the changes were mostly insignificant. After discontinuation of exposure the activities of enzymes and the concentrations of cytochromes returned to the control level in 1-4 days. The results obtained resemble the time-courses for the induction of monooxygenases by other inducers. The tissue differences suggest the unequal distribution of various cytochrome P-450 forms and their individual responsiveness to induction in liver, kidneys and lungs.     

Effects of thyroidectomy of the rat on the structure and functions of skeletal muscle mitochondria]

Mitochondria used in the present study were isolated from skeletal muscle of normal and thyroidectomized rats. The preparations were controlled by electron microscopy. It was not possible to find any morphological change induced by thyroidectomy, nevertheless, some difference appeared in the cytochrome contents which were slightly decreased. Oxygen consumption rates of thyroidectomized rat mitochondria were decreased when the particles were maintained in states 3 and 4 in the presence of various substrates, but the P/O ratios were not modified. The activities of mitochondrial enzymes were in general slightly affected by thyroidectomy except for glycerol-1-phosphate cytochrome c reductase and NADH rotenone sensitive cytochrome c reductase which were decreased and for glutamate dehydrogenase activity which was increased. The tRNA nucleotidyltransferase activity found in the mitochondrial matrix was not influenced by the absence of thyroid secretion. Normal rat muscle mitochondria incorporate 14C-leucine with an artificial ATP-generating system or with a respiratory substrate. The amino acid incorporation was decreased by thyroidectomy. Muscle mitochondria analyzed by polyacrylamide gel electrophoresis contained more than 30 protein components with MW ranging from 10.000 to 135.000. Thyroidectomy lowered the amount of a fraction of about 54.000 MW. It is not impossible that all the data observed in the absence of thyroid secretion are in relation with changes induced in the mitochondrial genome as previously shown in mitochondria isolated from liver or thyroidectomized rats.