In Vitro Study of Mitochondrial Protein Synthesis during Mitochondrial Biogenesis in Excised Plant Storage Tissue

Mitochondrial biogenesis was induced in Jerusalem artichoke (Helianthus tuberosus) tuber by aging tissue discs in distilled water for up to 26 hours. Changes in the purified mitochondrial fraction during aging included an increase in both protein content and specific respiratory activity. Using intact isolated mitochondria, conditions were optimized for incorporation of radioactive amino acid into protein. Incorporation was dependent upon the supply of an oxidizable substrate or an external ATP-generating system and showed characteristic sensitivity to inhibitors of protein synthesis. Aging of the tissue resulted in a 3-fold increase in the rate of in vitro incorporation of [³⁵S]methionine into mitochondrial protein. An analysis of the free amino acid pool in the mitochondrial fraction showed that the decrease in methionine level during aging of intact tissue was sufficient to account for the increased rate of protein labeling. The activation of mitochondrial biogenesis which occurs after slicing is not dependent on an increase in the capacity of mitochondria to synthesize protein as assayed in vitro.     

Increase of Mitochondrial Fraction in Sweet Potato Root Tissue after Wounding or Infection with Ceratocystis fimbriata

The acid-insoluble nitrogen content, lipid content, and cytochrome oxidase activity in the mitochondrial fraction are found to increase during incubation of slices of sweet potato (Ipomoea batatas) root tissue. These increases appear to be related to an increase in the number of the mitochondrial particles. The increase in the mitochondrial fraction is not accompanied by an increase in cell number. The nitrogen content in the mitochondrial fraction increases prior to the changes in the activity of cytochrome oxidase and lipid content. The increase in the numbers of the mitochondrial particles lags behind the increase in the cytochrome oxidase activity. Such findings are also found in the tissue infected by Ceratocystis fimbriata.     

Proteomic DIGE analysis of the mitochondria‐enriched fraction from aged rat skeletal muscle

Skeletal muscle aging is associated with a loss in tissue mass and contractile strength, as well as fiber type shifting and bioenergetic adaptation processes. Since mitochondria represent the primary site for energy generation via oxidative phosphorylation, we investigated potential changes in the expression pattern of the mitochondrial proteome using the highly sensitive DIGE approach. The comparative analysis of the mitochondria‐enriched fraction from young adult versus aged muscle revealed an age‐related change in abundance for 39 protein species. MS technology identified the majority of altered proteins as constituents of muscle mitochondria. An age‐dependent increase was observed for NADH dehydrogenase, the mitochondrial inner membrane protein mitofilin, peroxiredoxin isoform PRX‐III, ATPase synthase, succinate dehydrogenase, mitochondrial fission protein Fis1, succinate‐coenzyme A ligase, acyl‐coenzyme A dehydrogenase, porin isoform VDAC2, ubiquinol‐cytochrome c reductase core I protein and prohibitin. Immunoblotting, enzyme testing and confocal microscopy were used to validate proteomic findings. The DIGE‐identified increase in key mitochondrial elements during aging agrees with the concept that sarcopenia is associated with a shift to a slower contractile phenotype and more pronounced aerobic‐oxidative metabolism. This suggests that mitochondrial markers are reliable candidates that should be included in the future establishment of a biomarker signature of skeletal muscle aging.     

Endogenous elemental sulfur (S°) in dormant and aging α-spores of Phomopsis viticola

Endogenous elemental sulfur (S°) was measured in dormant α-spores of Phomopsis viticola Sacc. (ATCC 44940) from young (25-day-old) and aging (105-day-old) cultures grown on malt extract agar medium enriched with [³⁵S]-MgSO₄. Endogenous S° from the mitochondrial fraction, and the lipid and aqueous cytoplasmic fractions of young and aging α-spores were purified by column chromatography followed by thin-layer chromatography. The purity of mitochondrial pellets were checked by the catalase (EC 1.11.1.6) and acid phosphatase (EC 3.1.3.1) activities. Activities of the mitochondrial enzymes NAD⁺-isocitrate dehydrogenase (EC 1.1.1.41) and cytochrome c oxidase (EC 1.9.3.1) were also measured to determine the distribution of the endogenous S° between mitochondria and cytoplasm. In young dormant α-spores, endogenous S° was mostly found in the cytoplasmic lipid reserves, which were mainly phospholipids. The mitochondrial fraction of these young α-spores contained ca 10% of the total endogenous S°, whereas in aging α-spores stored for 105 days the endogenous S° was mainly (ca 90%) localized in the mitochondrial fraction. This accumulation of S° in mitochondria of aging α-spores was correlated with a sharp decrease in phospholipid reserves, endogenous and exogenous respiratory activities, ATP concentration, uptake of sulfate, and NAD⁺-isocitrate dehydrogenase and cytochrome c oxidase activities. These metabolic changes were correlated with an irreversible loss of germination capacity which leads to the natural death of P. viticolaα-spores. During the first min of the breaking of dormancy, the young α-spores possess a 7.3-fold capacity to reduce exogenous S° with production of hydrogen sulfide, as compared to the aging α-spores. In young α-spores the production of hydrogen sulfide was almost totally inhibited by 40 μM antimycin A (92%), and strongly inhibited by 2mM azide (75%) and by 15 μM 2,4-dinitrophenol (63%). Our work suggests that endogenous S° plays a key role in the regulation of the dormancy and aging processes of α-spores of P. viticola.     

Aging shifts mitochondrial dynamics toward fission to promote germline stem cell loss

Changes in mitochondrial dynamics (fusion and fission) are known to occur during stem cell differentiation; however, the role of this phenomenon in tissue aging remains unclear. Here, we report that mitochondrial dynamics are shifted toward fission during aging of Drosophila ovarian germline stem cells (GSCs), and this shift contributes to aging‐related GSC loss. We found that as GSCs age, mitochondrial fragmentation and expression of the mitochondrial fission regulator, Dynamin‐related protein (Drp1), are both increased, while mitochondrial membrane potential is reduced. Moreover, preventing mitochondrial fusion in GSCs results in highly fragmented depolarized mitochondria, decreased BMP stemness signaling, impaired fatty acid metabolism, and GSC loss. Conversely, forcing mitochondrial elongation promotes GSC attachment to the niche. Importantly, maintenance of aging GSCs can be enhanced by suppressing Drp1 expression to prevent mitochondrial fission or treating with rapamycin, which is known to promote autophagy via TOR inhibition. Overall, our results show that mitochondrial dynamics are altered during physiological aging, affecting stem cell homeostasis via coordinated changes in stemness signaling, niche contact, and cellular metabolism. Such effects may also be highly relevant to other stem cell types and aging‐induced tissue degeneration.     

Aging and Mitochondrial Development in Potato Tuber Tissue

Respiratory activity of mitochondria isolated from fresh and aged potato tuber tissue has been determined. No significant change in activity of the particles (expressed per unit N) was observed as a result of aging. However, the yield of mitochondrial material increased with aging. It has been suggested that the commonly observed stimulation of respiration that accompanies aging of potato tissue can be attributed largely to an inerease in the mitochondrial population rather than to a stimulation of the particles per se, and that this increase results from a fission of pre-existing mitochondria. The respiratory activity of mitochondria isolated from unaged tissue was found to decline following storage of the tubers in the cold for extended periods. This decline was attributed to a loss of cristae structure in the particles, as observed microscopically. The possibility that this reduction in structural organization might be associated with changes in the state of dormancy of the tissue was considered.     

Voluntary exercise normalizes the proteomic landscape in muscle and brain and improves the phenotype of progeroid mice

The accumulation of mitochondrial DNA (mtDNA) mutations is a suspected driver of aging and age‐related diseases, but forestalling these changes has been a major challenge. One of the best‐studied models is the prematurely aging mtDNA mutator mouse, which carries a homozygous knock‐in of a proofreading deficient version of the catalytic subunit of mtDNA polymerase‐γ (PolgA). We investigated how voluntary exercise affects the progression of aging phenotypes in this mouse, focusing on mitochondrial and protein homeostasis in both brain and peripheral tissues. Voluntary exercise significantly ameliorated several aspects of the premature aging phenotype, including decreased locomotor activity, alopecia, and kyphosis, but did not have major effects on the decreased lifespan of mtDNA mutator mice. Exercise also decreased the mtDNA mutation load. In‐depth tissue proteomics revealed that exercise normalized the levels of about half the proteins, with the majority involved in mitochondrial function and nuclear–mitochondrial crosstalk. There was also a specific increase in the nuclear‐encoded proteins needed for the tricarboxylic acid cycle and complex II, but not in mitochondrial‐encoded oxidative phosphorylation proteins, as well as normalization of enzymes involved in coenzyme Q biosynthesis. Furthermore, we found tissue‐specific alterations, with brain coping better as compared to muscle and with motor cortex being better protected than striatum, in response to mitochondrial dysfunction. We conclude that voluntary exercise counteracts aging in mtDNA mutator mice by counteracting protein dysregulation in muscle and brain, decreasing the mtDNA mutation burden in muscle, and delaying overt aging phenotypes.     

Presence of an inactive protein immunologically analogous to cytochrome c oxidase in the inner membrane of sweet potato root mitochondria

A protein, which was immunoreactive to antibody against cytochrome c oxidase, was found in the mitochondrial membrane fraction of sweet potato root tissue. The protein was associated relatively weakly with the mitochondrial inner membrane as compared with cytochrome c oxidase. It exerted no cytochrome c oxidase activity and contained no heme a. The protein was purified by phenyl-Sepharose column chromatography and polyacrylamide gel electrophoresis. The molecular weight of its polypeptide chain was 57,000. In addition, the protein decreased during aging of tissue slices. It is therefore not improbable that the protein is a precursor of cytochrome c oxidase composed of only the subunits of cytoplasmic origin, since aging of tissue slices has been shown to result in an increase in the enzyme activity which is inhibited by chloramphenicol but not by cycloheximide.     

Calcium containing particles in mitochondria of heart muscle cells as shown by cryo-ultramicrotomy and X-ray microanalysis

Summary Mitochondria of normal myocardial cells of the sand rat and the mouse as well as of the left ventricle of man, have been examined for their content of calcium. Ultrahistochemistry and X-ray microanalysis revealed two basically different inclusions: Osmiophilic mitochondrial granules and Spherical mitochondrial particles. Osmiophilic mitochondrial granules were found in conventionally fixed and plastic embedded tissues as well as in cryosections of chemically fixed and sucrose infused tissues. Such granules lacked inert electron density and probably consisted mainly of unsaturated lipids. X-ray spectra obtained from these tissues revealed no peaks for calcium. Spherical mitochondrial particles were present in dry-cut cryo-sections of N₂-frozen tissues not treated by fixatives and/or cryoprotectants. These particles were deeply electron dense in unstained, freeze-dried cryo-sections. They usually measured from 600Å–900Å in diameter in the normal myocardium of the sand rat and the mouse and from 250 Å–400Å in diameter in the left ventricular myocardium of man. Significant calcium peaks could be identified in the X-ray spectra of these particles, whereas none occurred in the analyses of other tissue regions. Potassium was detected with about equal frequency in the particles and in other parts of the tissue. On the basis of the inert electron density of the particles and their absence in chemically fixed tissues as well as of the results of the X-ray analysis, it is concluded that they contain precipitates of extremely labile ions of mitochondrial calcium.We should like to thank Mrs. Trine Jensen for skillful technical assistance. This work was supported by grants from The Norwegian Council on Cardiovascular Disease and from the Norwegian Research Council for Science and the Humanities     

Growth differentiation factor 15 protects against the aging‐mediated systemic inflammatory response in humans and mice

Mitochondrial dysfunction is associated with aging‐mediated inflammatory responses, leading to metabolic deterioration, development of insulin resistance, and type 2 diabetes. Growth differentiation factor 15 (GDF15) is an important mitokine generated in response to mitochondrial stress and dysfunction; however, the implications of GDF15 to the aging process are poorly understood in mammals. In this study, we identified a link between mitochondrial stress‐induced GDF15 production and protection from tissue inflammation on aging in humans and mice. We observed an increase in serum levels and hepatic expression of GDF15 as well as pro‐inflammatory cytokines in elderly subjects. Circulating levels of cell‐free mitochondrial DNA were significantly higher in elderly subjects with elevated serum levels of GDF15. In the BXD mouse reference population, mice with metabolic impairments and shorter survival were found to exhibit higher hepatic Gdf15 expression. Mendelian randomization links reduced GDF15 expression in human blood to increased body weight and inflammation. GDF15 deficiency promotes tissue inflammation by increasing the activation of resident immune cells in metabolic organs, such as in the liver and adipose tissues of 20‐month‐old mice. Aging also results in more severe liver injury and hepatic fat deposition in Gdf15‐deficient mice. Although GDF15 is not required for Th17 cell differentiation and IL‐17 production in Th17 cells, GDF15 contributes to regulatory T‐cell‐mediated suppression of conventional T‐cell activation and inflammatory cytokines. Taken together, these data reveal that GDF15 is indispensable for attenuating aging‐mediated local and systemic inflammation, thereby maintaining glucose homeostasis and insulin sensitivity in humans and mice.     

Mitochondrial Activity in Orobanche cernua from Different Regions of the Scape and as Influenced by Host

Orobanche cernua, a holoparasite, was harvested from different hosts, namely, Solanum melongena, Petunia hybrida, Lycopersicum esculentum, Solanum nigrum and Datura metel. Mitochondrial particles were isolated and they were evaluated in terms of the marker enzyme, cytochrome c oxidase, and protein in the mitochondrial fraction. Protein levels in whole homogenate and mitochondrial fraction of parasite growing on different host plants did not vary significantly, whereas the recovery of protein (% of whole homogenate) in the mitochondrial fraction of parasite growing on D. metel was higher. Cytochrome c oxidase activity in parasites growing on the three host plants varied, being highest when the parasite grew on S. melongena, followed sequentially by that on P. hybrida and D. metel. Protein in whole homogenate and mitochondrial fraction was significantly lower, 25–36% and 15–33%, respectively, in distal region when compared with the proximal region. Similarly, cytochrome oxidase and respiratory activity was significantly lower, 23–34% and 18–23%, respectively, in the distal region of the parasite. In addition, variations in cytochrome oxidase and respiratory activity in the proximal and distal regions of the parasite growing on different hosts was also significant. Results indicated that mitochondria in haustoria‐bearing proximal region of Orobanche scape play a special role to meet the metabolic demand of the parasite.     

Effects of Particle Size and Redox Potential on Arsenic Fractionation in Soils Irrigated with Arsenate-rich Water

Aging affects arsenic (As) bioaccessibility in soils. This study focuses on the influences of particle size and redox potential on As(V) aging in irrigated soils. The results showed that variation of As fractions in fine particles, except the loosely adsorbed fraction, was larger than that in coarse particles over time. Anoxic conditions decreased the change in As fractions, with the exception of the exchangeable fraction in soils over time, in comparison to the aerobic condition. The aging processes of As(V) in different particle sizes and soils at different redox potentials exhibited several stages. The only significant difference in the aging process of As(V) in different particle sizes was the longer transformation period of the water-soluble fraction into the Fe/Mn/Al oxides-bound fraction in fine particles than in coarse particles. The redox potential had a significant influence on the aging process of As(V) in soils after 10 days of incubation. In terms of As bioaccessibility, anoxic conditions shortened the aging process of As(V) in soils. During the aging process, fine particles and aerobic conditions intensified the decrease in As(V) bioaccessibility in soils in comparison to the coarse particles and anoxic condition.     

Size-Dependent Neurotoxicity of Aluminum Oxide Particles: a Comparison Between Nano- and Micrometer Size on the Basis of Mitochondrial Oxidative Damage

Aluminum nanoparticles (AlNPs) are among the most abundantly produced nanosized particles in the market. There is limited information about the potential harmful effects of aluminum oxide due to its particle size on human health. Considering the toxic effects of Al on brain as its target tissue, in this study, the toxicity of nanoparticles, microparticles, and ionic forms of Al on rat brain and isolated mitochondria was evaluated. Sixty male Wistar rats were divided into ten groups (six rats each), in which group I was the control, and the other groups were administered different doses of Al nanoparticles, Al microparticles (AlMP), and Al ionic forms (2, 4, and 8 mg/kg, i.p.) for 28 days. After 24 h, the animals were killed, brain tissue was separated, the mitochondrial fraction was isolated, and oxidative stress markers were measured. Also, mitochondrial function was assayed by MTT test. The results showed that all forms of Al particles induced ROS formation, lipid peroxidation, protein oxidation, glutathione depletion, mitochondrial dysfunction, and gait abnormalities in a dose-dependent manner. In addition, Al particles decreased mitochondrial membrane potential. These data indicated that oxidative stress might contribute to the toxicity effects of Al. Comparison of oxidative stress markers between all forms of Al revealed that the toxic effect of AlNP on brain tissue was substantially more than that caused by AlMP and bulk form. This study showed more neurotoxicity of AlNPs compared to other forms on brain oxidative damage that probably is due to more penetration into the brain.     

Changes in energy status of leaf cells as a consequence of mitochondrial genome rearrangement

The MSC16 cucumber (Cucumis sativus L.) mutant with lower activity of mitochondrial Complex I was used to study the influence of mitochondrial metabolism on whole cell energy and redox state. Mutant plants had lower content of adenylates and NADP(H) whereas the NAD(H) pool was similar as in wild type. Subcellular compartmentation of adenylates and pyridine nucleotides were studied using the method of rapid fractionation of protoplasts. The data obtained demonstrate that dysfunction of mitochondrial respiratory chain decreased the chloroplastic ATP pool. No differences in NAD(H) pools in subcellular fractions of mutated plants were observed; however, the cytosolic fraction was highly reduced whereas the mitochondrial fraction was more oxidized in MSC16, as compared to WTc. The NADP(H) pool in MSC16 protoplasts was greatly decreased and the chloroplastic NADP(H) pool was more reduced, whereas the extrachloroplastic pool was much more oxidized, than in WTc protoplast. Changes in nucleotides distribution in cucumber MSC16 mutant were compared to changes found in tobacco (Nicotiana sylvestris) CMS II mitochondrial mutant. In contrast to MSC16 cucumber, the content of adenylates in tobacco mutant was much higher than in tobacco wild type. The differences were more pronounced in leaf tissue collected after darkness than in the middle of the photoperiod. Results obtained after tobacco protoplast fractionating showed that the increase in CMS II adenylate content was mainly due to a higher level in extrachloroplast fraction. Both mutations have a negative effect on plant growth through perturbation of chloroplast/mitochondrial interactions.     

Heterogeneity of rat liver mitochondrial fractions and the effect of tri-iodothyronine on their protein turnover

1. Rat liver mitochondria were separated into heavy, light and fluffy fractions by differential centrifugation under standard conditions. 2. All mitochondrial fractions possessed soluble as well as membrane-bound enzymes typical of mitochondria. 3. The heavy fraction represented the stable mitochondrial structures and the fluffy particles appear to be loosely coupled. 4. The light mitochondrial fraction lacked the ability of coupled phosphorylation. 5. A study of mobility and isoelectric pH indicated a similarity in the basic membrane structure of all the mitochondrial fractions. 6. The turnover rates of proteins in the heavy and fluffy particles were almost identical; however, this rate was rapid for the light mitochondrial fraction. 7. On treatment with 3,3',5-tri-iodo-l-thyronine, succinoxidase activity was maximally stimulated much earlier in the light mitochondrial fraction than in the heavy fraction. The activity of the fluffy particles, however, remained almost unaffected. 8. Malate dehydrogenase activity in all the mitochondrial fractions was stimulated only at 40h after tri-iodothyronine treatment. 9. The pattern of incorporation of dl-[1-(14)C]leucine in vivo in the tri-iodothyronine-treated animals indicated a rapid initial incorporation and high synthetic ability of the light mitochondrial fraction. 10. The turnover pattern of proteins of the mitochondrial fractions from animals receiving repeated doses of tri-iodothyronine was remarkably different from the normal pattern and suggested that preformed soluble protein units may be incorporated in the light mitochondrial fraction during maturation to form the stable heavy mitochondria. 11. The amount of light-mitochondrial proteins decreased by 40% on thyroidectomy and increased by 160% on treatment with tri-iodothyronine. 12. The possible significance of these results is discussed in relation to mitochondrial genesis.     

Adenosine triphosphate:creatine phosphotransferase of rat cerebral cortex: evidence for a bimodal subcellular localization

—(1) ATP: creatine phosphotransferase of rat cerebral cortex is soluble to the extent of 57 per cent when the tissue is homogenized in 0.25 M-sucrose and 80 per cent when distilled water is used for tissue dispersion. Among particulate fractions, the crude mitochondria] fraction contains the highest percentage of enzyme activity. (2) Discontinuous sucrose gradient fractionation of the crude mitochondrial fraction yields about 55 per cent of the particulate activity in the nerve ending fractions and 24 per cent in the mitochondrial pellet. (3) Rupturing of the nerve-ending particles by a moderate osmotic shock designed to spare the mitochondria results in about 60 per cent of the ATP:creatine phosphotransferase becoming soluble, the remainder preserving the association with heavy particles, presumably mitochondria. (4) Subfractionation of the microsomal fraction on a discontinuous sucrose gradient reveals that this particulate component of the enzyme is an adsorption artifact. (5) The overall evidence points to at least two distinct subcellular localizations of the enzyme in rat brain cortex, a major soluble component and a particulate component. It has not been unequivocally shown whether the latter, in turn, reflects the presence of a single, mitochondrial component or whether the soluble matrix of the nerve ending particles represents a third locale for the enzyme.     

Methylene blue alleviates nuclear and mitochondrial abnormalities in progeria

Hutchinson–Gilford progeria syndrome (HGPS), a fatal premature aging disease, is caused by a single‐nucleotide mutation in the LMNA gene. Previous reports have focused on nuclear phenotypes in HGPS cells, yet the potential contribution of the mitochondria, a key player in normal aging, remains unclear. Using high‐resolution microscopy analysis, we demonstrated a significantly increased fraction of swollen and fragmented mitochondria and a marked reduction in mitochondrial mobility in HGPS fibroblast cells. Notably, the expression of PGC‐1α, a central regulator of mitochondrial biogenesis, was inhibited by progerin. To rescue mitochondrial defects, we treated HGPS cells with a mitochondrial‐targeting antioxidant methylene blue (MB). Our analysis indicated that MB treatment not only alleviated the mitochondrial defects but also rescued the hallmark nuclear abnormalities in HGPS cells. Additional analysis suggested that MB treatment released progerin from the nuclear membrane, rescued perinuclear heterochromatin loss and corrected misregulated gene expression in HGPS cells. Together, these results demonstrate a role of mitochondrial dysfunction in developing the premature aging phenotypes in HGPS cells and suggest MB as a promising therapeutic approach for HGPS.     

TYROSINE HYDROXYLASE IN BOVINE CAUDATE NUCLEUS

Approximately 80 per cent of tyrosine hydroxylase activity in bovine caudate nucleus was particle-bound. The rest of the activity was found in the soluble fraction. The enzyme activity in crude tissue preparations was inhibited, probably by the presence of endogenous inhibitors. Dilution of crude tissue preparations such as the crude mitochondrial fraction caused an increase in the specific activity. The particle-bound enzyme was solubilized by incubation with trypsin. The presence of deoxycholate increased the degree of solubilization. The activity of the solubilized enzyme from the washed particles was also inhibited, but the subsequent purification by ammonium sulphate could eliminate the inhibition. The solubilized enzyme was partially purified by ammonium sulphate fractionation and Sephadex G-150 chromatography. A tetrahydropteridine and ferrous ion were required as cofactors for the partially purified enzyme. Among various divalent cations, only ferrous ion could activate the partially purified enzyme. The enzyme was inhibited by L-α-methyl-p-tyrosine and catecholamines such as dopamine. The optimum pH was found between 5.5 and 6.0. K_m values toward tyrosine, 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine and Fe²⁺, were approximately 5 × 10^?5 M, 1 × 10^?4 M and 4 × 10^?4 M, respectively.     

Aging-related decrease in hepatic cytochrome oxidase of the Fischer 344 rat

The effect of aging on the hepatic mitochondrial population has been determined using a rigorously defined group of Fischer 344 rats with known survivorship data. The age groups studied included mature adult controls (8.5 months; 100% survivorship), an intermediate aged group (17.5 months; 90% survivorship), and an aged group (29 months; 20% survivorship). Cytochrome oxidase activity and content were determined in homogenates and mitochondrial fractions. The mitochondrial fractions were characterized by determination of respiratory activity, and monoamine oxidase activity as well as evaluation of the polypeptide composition by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis. The yield of protein in the isolated mitochondrial fraction as well as the mitochondrial specific content decreased significantly as a function of aging. Mitochondrial specific content was determined from the specific activities of cytochrome oxidase in the homogenate (per gram liver) and in the isolated mitochondrial fraction (per mg protein). Specific activity of hepatic cytochrome oxidase decreased approximately 15% (P = 0.035) in homogenates from the 17.5-month animals with a further, highly significant (P = 0.0002) decrease (29%) in the 29-month animals. In contrast, there was no statistically significant difference among the age groups in the cytochrome oxidase specific activity in the isolated hepatic mitochondrial fractions. However, the percentage of the total homogenate cytochrome oxidase activity recovered in the isolated mitochondrial fraction decreased significantly in the 29-month animals (P = 0.0063 vs the 8.5-month controls; P = 0.022 vs the 17.5-month group). Cytochrome aa₃ content of total liver homogenates from aged animals decreased (P = 0.00064) which is in agreement with the decline in cytochrome oxidase specific activity in this age group. In the mitochondrial fraction from the aged animals, cytochrome aa₃ content was essentially unchanged which is consistent with the lack of aging-related change in mitochondrial cytochrome oxidase specific activity. In freshly isolated mitochondrial fractions, no aging-related alterations were observed in respiratory control and $\text{ADP}\text{O}$ ratios. The addition of exogenous NADH and cytochrome c did not change significantly the respiratory rate of hepatic mitochondria from control or aged animals. These results demonstrate the integrity of freshly isolated mitochondrial preparations from both control and aged Fischer 344 rats. In addition, there was no aging-related alteration in either monoamine oxidase specific activity or polypeptide composition. The similarities observed in the specific activities of cytochrome oxidase and monoamine oxidase, as well as in the cytochrome aa₃ content and polypeptide composition of the isolated mitochondrial fraction, suggest a generalized decrease in hepatic mitochondrial content as a function of aging rather than a selective loss of mitochondrial components.