Specific Activity Of Cytochrome Oxidase Research Articles

Mitochondrial Dysfunction in EAE Mice Brains and Impact of HIF1-α Induction to Compensate Energy Loss

Background: Mitochondrial dysfunction may be involved in the process of degradation and death of gray matter cells of the central nervous system (CNS) in patients with multiple sclerosis (MS). MS is known as a chronic, progressive demyelinating disease of the CNS. Objectives: Experimental autoimmune encephalomyelitis (EAE) mouse model of MS is the best method for extracting data trend for diagnosing this disorder. The aim of this study was to evaluate the specific activity of the Cytochrome oxidase (COX), ATP, and hypoxia-inducible factor 1 alpha (HIF-1α) in brain tissues of the EAE mice model. Methods: Twenty-one female mice (C57BL/6) were used, 9 for inducing the EAE model and 6 for each of both negative and sham control groups. The specific activity of the COX, ATP, and HIF-1α levels were evaluated in the whole brain of all 3 mice groups. Results: According to the findings, specific COX activity and ATP levels were decreased significantly, which could be due to the mitochondrial dysfunction and neuronal loss in MS lesions, whereas HIF-1α levels increased significantly in the EAE mice group, compared to the sham and negative control groups. The significant increase of HIF-1α levels reinforces the hypothesis that the HIF-1α induction may provide prevention of neuronal death by compensating energy loss under hypoxia-like conditions in EAE mice brains. Conclusions: The results of this study suggest that HIF-1α induction may also be a potential target for controlling the progression of MS, or the development of HIF-1α inducing compounds could be a potential candidate for the management of this disease and provide a rationale to conduct further research in this area.

Higher platelet cytochrome oxidase specific activity in surviving than in non-surviving septic patients.

IntroductionIn a previous study with 96 septic patients, we found that circulating platelets in 6-months surviving septic patients showed higher activity and quantity of cytochrome c oxidase (COX) normalized by citrate synthase (CS) activity at moment of severe sepsis diagnosis than non-surviving septic patients. The objective of this study was to estimate whether COX specific activity during the first week predicts 1-month sepsis survival in a larger cohort of patients.MethodsUsing a prospective, multicenter, observational study carried out in six Spanish intensive care units with 198 severe septic patients, we determined COX activity per proteins (COXact/Prot) in circulating platelets at day 1, 4 and 8 of the severe sepsis diagnosis. Endpoints were 1-month and 6-months mortality.ResultsSurvivor patients (n = 130) showed higher COXact/Prot (P < 0.001) than non-survivors (n = 68) at day 1, 4 and 8 of severe sepsis diagnosis. More than a half of the 6-months survivor patients showed an increase in their COXact/Prot from day 1 to 8. However, most of the 1-month non-survivors exhibited a decrease in their COXact/Prot from day 1 to 8. Multiple logistic regression analyses showed that of platelet COXact/Prot > 0.30 mOD/min/mg at day 1 (P = 0.002), 4 (P = 0.006) and 8 (P = 0.02) was associated independently with 1-month mortality. Area under the curve of COXact/Prot at day 1, 4 and 8 to predict 30-day survival were 0.70 (95% CI = 0.63-0.76; P < 0.001), 0.71 (95% CI = 0.64-0.77; P < 0.001) and 0.71 (95% CI = 0.64-0.78; P < 0.001), respectively.ConclusionsThe new findings of our study, to our knowledge the largest series reporting data about mitochondrial function during follow-up in septic patients, were that septic patients that survive 1-month have a higher platelet cytochrome oxidase activity at moment of sepsis diagnosis and during the first week than non-survivors, and that platelet cytochrome oxidase activity at moment of sepsis diagnosis and during the first week could be used as biomarker to predict the clinical outcome in septic patients.

The Effect of Initial Moisture Content, Packaging and Storage Period on Succinate Dehydrogenase and Cytochrome Oxidase Activity of Soybean Seed

The quality of soybean seed is easily decreased, so that it is difficult to keep them for a long time. The research aims to study the effect of initial moisture content, packaging material and storage period on succinate dehydrogenase and cytochrome oxidase activity. The experimental design was arranged in Factorial in RCB consisting of 3 factors: moisture content, i.e. 8. 10 and 12 percent, packaging materials, i.e. polyethylene, wheat and aluminium foil; storage period i.e. 0, 1, 2, 3, 4, 5, and 6 months. Changes in seed water content, specific activity of succinate and cytochrome oxidase, respiration rate, germination coefficient velocity germination, and abnormal seedlings were monthly determined. It was found that seed moisture content was increase and showed positive correlation with seed moisture content and abnormal seedlings and showed negative correlation with specific activity of succinate dehydrogenase and cytochrome oxidase, respiratory rate, germination and coefficient velocity germination. From this experiment, soybean seeds were stored in polyethylene and aluminium foil bags observed highly succinate dehydrogenase and cytochrome oxidase specific activity, respiration rate, germination, coefficient velocity germination and keep moisture content in low level could delay seed deterioration followed by polyethylene and wheat bags. Key words: moisture content, succinate dehydrogenase, cytochrome oxidase, soybean

Biochemical and functional differences in rat liver mitochondrial subpopulations obtained at different gravitational forces

Previous studies have reported that liver mitochondria may be fractionated into different subpopulations. However, no careful studies have been performed to exclude mitochondrial damage and to investigate more thoroughly the possible biochemical differences existing between the tip. In this study, we analysed the integrity and the biochemical properties of rat liver a. Mitochondrial fractions were obtained by differential centrifugation at diffiereat gravitational forces: 1000 g (Ml fraction), 3000 g (M3 fraction) and 10,000g (M10 fraction). 11re integrity of these organelles was checked by measuring citrate syMhaae activity both in the presence and absence of Triton X-100 detergent. Biochemical analyses included po determination of cytochrome oxidame activity and respiratory parameters and spectrophotometric determination of cytochrome content. (1) The integrity of a was almost homogeneous between fractions (88.5, 80 and 78.3% in Ml, M3 and M10 fractions, respectively). (2) The heaviest Ml fraction contains mitocin which are on average twee as large as M3 and about three times as large as M10. (3) The Ml fraction exhibited the highest specific cytochrome oxidase activity (1040 ± 20 nAtoms O/min x mg protein) and the highest respiratory rates (72 ± 3 nAtoms O/min x mg protein and 526 ± 45 nAtoms O/min × m protein for States 4 and 3, respectively). Oxidative capacity and respiratory rates decreased as the size of the organelies decreased, reaching values of 1 5 and 1 14 in the M3 and M10 fractions as compared to the Ml. (4) These changes are accompanied by a change in the respiratory control ratio (RCR), which varies from 7.3 in Ml to about 2.0 in M10. A molar trend was observed in cytochrome contents but the differences were not as great as cytochrome oxidese activity and State 3 respiration. These results, as a whole, show that a mitechondrini heterogeneity exists in rat liver cell. We suggest that the above-memioned differences might represent step of mkocbondrW maturation. The maturation would be fundamentally based on the increase of efficiency of the mechanism for ATP synthesis.

Isolation and physiological properties of intact mitochondria from bell pepper ( Capsicum annum) fruit

Summary A protocol for isolating mitochondria from bell pepper (Capsicum annum, var. ‹Jupiter›) fruit by differential centrifugation and purification on a discontinuous 2-step Percoll gradient was developed based on modifications of techniques employed for other plant tissues. The mitochondrial fraction banded at the interface of the two Percoll concentrations, and was well separated from thylakoid fragments. Percoll-purified mitochondria exhibited high oxidative and phosphorylative capacities with high respiratory control and ADP/O ratios. Catalase activity decreased during purification of mitochondria, with levels associated with purified mitochondria representing only about 3% of those present in crude mitochondrial fractions. The specific activity of cytochrome oxidase increased during purification. Membrane competence of the purified mitochondria was about 90 %, and the typical double-membrane system remained otherwise intact as revealed in electron micrographs. Mitochondrial ATPase (α-subunit) and alternative oxidase proteins were detected at typical molecular-mass values, providing additional evidence for the maintenance of mitochondrial membrane integrity during the purification process. Bell pepper fruit mitochondria are physiologically competent in vitro and should prove useful in studies of the respiratory metabolism of fruit ripening and senescence.

The regulation of oxidative phosphorylation in plant mitochondria: The roles of the quinone-oxidizing and -reducing pathways

Introduction I n eukaryotic cells, mitochondrial function to generate both A7'P (via oxidative phosphorylation) and carbon skeletons (via the tricarboxylic acid cycle) for biosynthetic purposes. Although the cytosolic demands for A T P can be met through the activity of the mitochondrial ATP synthetase in non-green cells, the situation is more complex in photosynthetic cells, since here the supply of cytosolic A'I'I' can be met by photophosphorylation through the indirect transfer of ATP from the chloroplast stronia. IJnlike their mammalian counterpart, the metabolic role of plant mitochondria appears to be developmentally regulated, and distinct biochemical and morphological changes have been demonstrated to occur to mitochondria as the plant cell matures and develops photosynthetically [ 11. Such changes appear to be dependent upon a number of factors including cell maturity, tissue location and the interaction with other organelles such as chloroplasts and peroxisornes. For instance, in the basal meristematic cells of monocotyledons (such as wheat or barley) the specific activities of cytochrome oxidase and of succinate dehydrogenase are relatively high and reach a constant level at and above 4cm from the base of the leaf (21 where glycine decarboxylase (a key enzyme in the photorespiratory carbon cycle) increases substantially above this level [ 3 , 41. Morphometric analysis also indicates that the number of mitochondria per mesophyll cell increases between the basal region of the leaf and the region 1.5 cm above it, but above this region mitochondrial numbers either remain constant or decrease [ 11. Similarly the relative abundance of the a-subunit of the ATP synthetase remains relatively constant along the length of the leaf [ 51. All these changes probably reflect the changing metabolic function of mitochondria as the plant cell develops. I n tissues with a high metabolic rate, such as the areas involved in cell division and in expansion, the production of ATI' is a key metabolic role of mitochondria. but as the cell matures ;ind becomes photosynthetically active, the mitochondrial function changes t o a more biosynthetic role in providing carbon skeletons. In leaf tissue, the development of photosynthesis and the associated

Effect of 3,3′-di-iodothyronine and 3,5-di-iodothyronine on rat liver mitochondria

In the present study we report that 3,3',5-tri-iodothyronine (T3) as well as two iodothyronines (3,5-di-iodothyronine (3,5-T2) and 3,3'-di-iodothyronine (3,3'-T2)) significantly influence rat liver mitochondrial activity. Liver oxidative capacity (measured as cytochrome oxidase activity/g wet tissue) in hypothyroid compared with normal rats was significantly reduced (21%, P > 0.01) and the administration of T3 and both iodothyronines restored normal values. At the mitochondrial level, treatment with T3 stimulated respiratory activity (state 4 and state 3) and did not influence cytochrome oxidase activity. On the other hand, both the mitochondrial respiratory rate and specific cytochrome oxidase activity significantly increased in hypothyroid animals after treatment with 3,3'-T2 or 3,5-T2 (about 50 and 40% respectively). The actions of both iodothyronines were rapid and evident by 1 h after the injection. The hepatic mitochondrial protein content which decreased in hypothyroid rats (9.6 mg/g liver compared with 14.1 in normal controls, P < 0.05) was restored by T3 injection, while neither T2 was able to restore it. Our results suggest that T3 and both iodothyronines have different mechanisms of action. T3 acts on both mitochondrial mass and activity; the action on mitochondrial activity was not exerted at the cytochrome oxidase complex level. The action of the iodothyronines, on the other hand, is exerted directly on the cytochrome oxidase complex without any noticeable action on the mitochondrial mass.

Nonshivering thermogenesis in king penguin chicks. II. Effect of fasting.

The effect of fasting on the energy metabolism of skeletal muscle and liver was investigated in cold-acclimatized short-term fasting (STF) (3 wk) and naturally long-term fasting (LTF) (4-5 mo) king penguin chicks, both groups exhibiting nonshivering thermogenesis (NST). A comparison was made with nourished cold-acclimatized controls. In these chicks, no brown adipose tissue deposits could be found on electron-microscopic observations of fat deposits. Protein content and cytochrome oxidase (CO) activity of tissue homogenates were measured in liver and pectoralis and gastrocnemius muscles, as were protein content, CO activity, and respiration rates of mitochondria isolated from these organs. Fasting-induced protein loss affected the pectoralis more than the gastrocnemius muscle, thus preserving locomotor function. In STF chicks, specific mitochondrial protein content and specific tissue CO activity were preserved but total organ CO capacity was reduced by half in pectoralis and liver following the fall in organ mass. In LTF chicks, both specific and total CO activity were drastically reduced in muscles, whereas specific CO activity was preserved in liver. In these LTF chicks, muscle mitochondria showed an energized configuration associated with an increased area of inner membrane in gastrocnemius. A reduction of respiratory control ratio (RCR) was observed in subsarcolemmal muscle mitochondria of STF chicks, whereas intermyofibrillar and liver mitochondria kept high RCR values.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of chronic alcohol consumption on the steady-state kinetics properties of cytochrome oxidase in rat liver

The effect of chronic alcohol consumption on steady-state kinetic characteristics of cytochrome oxidase in rat liver was studied using submitochondrial particles prepared from ethanol-fed and control rats. Preparations from both control and alcoholic rats had equivalent apparent K m values for cytochrome c of 13 μM in the presence of phenazine methosulfate or 19 μM with N, N, N′, N′-tetramethylphenylene diamine as oxidation-reduction mediators at physiological ionic strength. Both preparations showed comparable stimulation (approx. 3-fold) of oxidase activity following detergent solubilization of the membrane and similar temperature dependence for oxidase activity. Under all conditions, preparations from alcohol-fed rats displayed 30 to 50% lower rats of cytochrome oxidase activity per unit membrane protein than those from control rats. The diminution in specific activity per mg protein was accompanied by a similar decline in heme aa 3 content, as has been noted in previous studies. When expressed on a turnover number basis, the molecular activity of cytochrome oxidase (natoms O / min per nmol heme a) was equivalent in both alcoholic and control preparations. The results indicate that the intrinsic kinetic characteristics of cytochrome oxidase are not changed by alcohol consumption. The data suggest that the characteristic decline in heme aa 3 content and cytochrome oxidase specific activity seen in ethanol-fed rats does not arise from alterations in the accessibility of the oxidase towards cytochrome c, or from changes in bulk phase lipid composition or physical properties. The results support the conclusion that ethanol consumption decreases the membrane content of functionally active oxidase molecules, but does not change the catalytic properties of these oxidase molecules.

Superoxide dismutase and cytochrome oxidase activities in light and heavy synaptic mitochondria from rat cerebral cortex during aging

The specific activities of superoxide dismutase (SOD) and cytochrome oxidase (COX) in light and heavy synaptic mitochondria from rat cerebral cortex at different ages (4, 8, 12, 16, 20, and 24 months) have been measured. The specific activity of COX was significantly higher in light synaptic mitochondria than in heavy ones at all ages examined. However, no significant difference during aging was shown. A marked decrease of SOD in light and heavy synaptic mitochondria during aging was observed.

Effect of aging and acetyl- l-carnitine on energetic and cholinergic metabolism in rat brain regions

The effect of aging and subchronic treatment with acetyl- l-carnitine (50 mg/kg per day) was studied on mitochondrial bioenergetics and cholinergic metabolism in non-synaptic mitochondria and synaptosomes isolated from cerebral cortex, hippocampus and striatum of rats aged 4, 11 and 18 months. Respiratory activity and cytochrome oxidase specific activity were unaffected by aging in non-synaptic mitochondria. In synaptosomes, pyruvate dehydrogenase, choline acetyltransferase and acetylcholinesterase specific activity remained unchanged, but the high-affinity choline uptake decreased in cerebral cortex and striatum of 18-month-old rats. Acetyl- l-carnitine treatment increased the high-affinity choline uptake in cerebral cortex of 18-month-old rats. The treatment caused also an increase in cytochrome oxidase activity in all the three cerebral regions and in choline uptake in the hippocampus, parameters that were not directly affected by aging processes.

Interacting effects of diet and environmental temperature on biochemical parameters in the liver ofLeuciscus idus melanotus (Cyprinidae: Teleostei)

One year old golden ide (Leuciscus idus melanotus) were starved for 2 weeks at 20°C (time zero) and then they were fed either freeze-dried mosquito larvae (natural diet) or a commercial fish chow (artificial diet) at an ambient temperature of 14°C and 20°C, respectively. Growth parameters and biochemical data in the liver were measured at time zero and after 3 and 7 weeks of specific regimen. If compared to natural food, the artificial diet caused transient increase in anabolic activity of the liver, but prevented long-lasting hepatocyte proliferation (increase of total liver DNA). After 3 weeks on artificial diet, the body weight was significantly higher and the liver mass doubled compared to ide kept on the natural regimen; tissue DNA content indicated that the hepatocyte volume increased mainly by increased protein content and corresponding uptake of water; lipid and glycogen contents were increased by a factor of 3-6, and the RNA∶DNA ratio was increased accordingly. The growth of the ide, as reflected by the condition factor and hepatosomatic index (HSI), was virtually stopped when the artificial regimen was maintained for 7 weeks, whereas on natural food the condition factor was increased and liver weight and DNA were doubled. Growth of the fish maintained at 14°C was significantly lower than that of fish maintained at 20°C, though the total liver DNA was still increased on the natural regimen during the experiment. Less lipid was stored by fish maintained at 14°C, whereas protein and glycogen deposits were enlarged, if compared to fish maintained at 20°C. The specific activity of cytochrome oxidase in liver mitochondria and of NADPH cytochrome c reductase in the microsomal fraction were found independent of diet and ambient temperature. Oxidative capacity of hepatocytes (mitochondrial protein/mg DNA) remained unchanged, and microsomal protein/cell appeared reduced in response to the artificial food. However, reduction of cell number on this diet resulted in less total mitochondrial and microsomal protein in the organ. Golden ide liver is found to be a sensitive experimental model to characterize the mutual influence of diet and temperature on fish; the results are discussed with regard to the usage of golden ide as test fish for water pollution.

Derivative spectrophotometric studies on cytotoxic effects of carbon monoxide

Characteristics of cytochrome oxidase prepared from hearts of Sprague—Dawley male rats were studied with the use of the fourth derivative spectrophotometry in respect to the cytotoxic effects of carbon monoxide (CO). CO-exposed rats tended to show lower specific activities of cytochrome oxidase than control and recovered rats. Moreover, there was a significant difference in the fourth derivative spectral features of the enzyme: CO-exposed groups indicated parks at 412 nm in the spectra while controls at 408 nm. This spectral difference seemed to reflect specific effect of CO on cytochrome oxidase, though such trace remained for not more than a day after death.

Structural alterations of the inner mitochondrial membrane in ischemic liver cell injury

Mitoplasts were prepared from 3-h ischemic livers in an attempt to define the structural alterations in the inner membrane that may account for the functional deficiencies of ischemic mitochondria. Mitoplasts from both control and ischemic livers had similar specific activities of cytochrome oxidase and succinate-cytochrome c reductase. With both preparations, the specific activity of rotenone-insensitive NADH-cytochrome c reductase was 10-fold lower than in the mitochondria from which they were prepared. Ischemic mitoplasts had no respiratory control with ADP, and had a slightly reduced phospholipid to protein ratio and an increased cholesterol to protein ratio. As a result, the cholesterol to phospholipid molar ratio was increased from the control of 0.04 to 0.08. There were also differences in the content of individual phospholipid species. Phosphatidylcholine increased by 15%, while cardiolipin decreased by 60%. There were increases in sphingomyelin and in the lysophospholipids of phosphatidylcholine, ethanolamine, and cardiolipin. Pretreatment with chlorpromazine did not prevent these changes. Linoleic acid was decreased by 35% in ischemic phospholipids, and the content of free fatty acids was increased 4-fold. Electron spin resonance spectroscopy of mitoplasts spin labeled with either 5- or 12-doxyl stearic acid revealed an increased molecular order (decreased fluidity) of ischemic inner mitochondrial membranes consistent with the increased cholesterol to phospholipid ratio. The data indicate activation of a phospholipase A in ischemic mitochondria with the resulting accumulation of products of lipid hydrolysis. This conclusion further emphasizes the close similarity between the structural and functional consequences of ischemia in the intact animal and the effect on isolated mitochondria of the activation of the endogenous phospholipase A. In both cases the major functional alterations are attributable to changes in the permeability of the inner mitochondrial membrane induced by the accumulation of lysophospholipids.

Leucine catabolism during the differentiation of 3T3-L1 cells. Expression of a mitochondrial enzyme system.

Leucine can be utilized efficiently as a precursor for lipid biosynthesis by adipose tissue, especially in the presence of glucose or glucose and insulin. During the differentiation of 3T3-L1 fibroblasts to adipocytes, the rate of lipid biosynthesis from L-[U-14C]leucine increases at least 30-fold and lipogenesis, with [U-14C] acetate as the precursor, increases by 10- to 15-fold. The specific activities of two mitochondrial dehydrogenases in the leucine oxidative pathway, the branched chain alpha-ketoacid dehydrogenase and isovaleryl-CoA dehydrogenase, as well as of leucine:alpha-ketoglutarate transaminase, increase at least 20-fold during the adipose conversion. Isovaleryl-CoA dehydrogenase was assayed in crude extracts using a specific fluorimetric method employing electron transfer flavoprotein as the electron acceptor for the flavoprotein dehydrogenase. The specific activity of 3-hydroxy-3-methylglutaryl-CoA lyase, the mitochondrial enzyme catalyzing the terminal reaction in the leucine degradation pathway, increases 4-fold during differentiation. The increases in the specific activities of the mitochondrial enzymes occur without a change in the specific activity of cytochrome oxidase, indicating that the increases do not simply reflect proliferation of mitochondria. The biosynthesis of at least 20 soluble mitochondrial polypeptides is enhanced during the adipose conversion of the fibroblasts as determined by polyacrylamide gel electrophoresis following incubation of the cells with [35S] methionine. The results provide a conservative estimate of the extent of changes in mitochondrial soluble proteins during the adipose conversion. They also establish that differentiated 3T3-L1 adipocytes metabolize leucine like mature adipose tissue and illustrate the roles of the branched chain alpha-ketoacid dehydrogenase and isovaleryl-CoA dehydrogenase in lipogenesis.

Oxygen-stimulated cytochrome oxidase assembly in hepatocyte monolayer cultures.

AbstractIsolated rat liver hepatocytes exhibited a twofold to threefold increase in both specific cytochrome oxidase activity and heme a concentration during their first 24 h in monolayer culture. Other mitochondrial respiratory enzyme activity did not increase over this same period. Decreasing the oxygen content of the gas phase to 5% of ambient eliminated the stimulation in heme a content without having an effect on cell protein synthesis or viability. These results indicate that oxygen concentration can effect the level of cytochrome oxidase (heme a) in these differentiated cells. The effect of oxygen may be on the translation or assembly of subunits, synthesis of heme, or incorporation of heme into holoenzyme. Oxygen stimulation of cytochrome oxidase content was not accompanied by increased incorporation of pulsed radiolabelled methionine into immunoprecipitated holoenzyme subunits. Moreover, chloramphenicol, a translation inhibitor of three mitochondrially synthesized cytochrome oxidase subunits, did not diminish the increase in heme a seen over the first 24 h of culture. These latter results suggest that the increasing enzyme content is not due to stimulated translation.

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 aa 3 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 aa 3 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 ADP 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 aa 3 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.

Mitochondrial number, cytochrome oxidase and succinic dehydrogenase activity in Xenopus laevis oocytes

An estimate has been made of the numbers of mitochondria in the mitochondrial cloud (Balbiani body) of Xenopus laevis oocytes ranging in size from 50 to 250 micrometers. The mitochondrial number is expressed in terms of a 'standard' organelle measuring 2 micrometers in length and 0.2 micrometer in diameter and is derived by measurements on electron micrographs of sections through the cloud. It is found that the amount of mitochondrial material rises very rapidly as the oocyte grows in size. At the time the cloud disperses, in oocytes of about 300 micrometers in diameter, it is estimated that there are the equivalent of over 500 000 mitochondria in each cell. The rate of increase is very similar to the rate of accumulation of mitochondrial DNA during the same period of growth. Using a polarographic technique the specific activity of cytochrome oxidase and succinic dehydrogenase was determined in mitochondrial fractions isolated from oocytes over a size range of 80-1200 micrometers in diameter. Although the specific activity of succinic dehydrogenase remains constant that of cytochrome oxidase falls sharply during the period when the mitochondria are replicating rapidly, i.e. up to about 300 micrometer diameter. In larger oocytes the specific activity of enzymes appears to remain constant but increasing contamination of the isolated mitochondrial fraction does not allow conclusions to be drawn from the enzyme loading of the mitochondria once they have dispersed from the cloud. The results are discussed in relation to the possibility that mitochondrial replication preceeds, or at least outpaces, mitochondrial differentiation during the course of oogenesis.

Inhibition of lysosomal function in red and white skeletal muscles by chloroquine

In chickens treated 7 days with chloroquine, morphological observations and chemical analyses were in agreement with our hypothesis that lysosomes degrade some fraction of skeletal muscle mitochondria, plasma membrane, and glycogen but apparently not normal myofibrillar proteins. After chloroquine inhibition of lysosomal digestion, autophagy was apparent in anterior (ALD) and posterior (PLD) latissimus dorsi muscles. Membrane-limited vacuoles (autophagic vacuoles) contained mitochondria, membranes, and glycogen. To help identify the vacuolar constituents, a variety of marker enzyme activities and chemical analyses were monitored for the ALD and PLD muscles. Parallel increases in 5′-nucleotidase and cytochrome oxidase specific activities correlated with the increase in observable inclusions. Autolytic rates were increased in acidic ( pH 4.0) but not in alkaline ( pH 8.5) or neutral ( pH 7.0) conditions when homogenates were prepared from muscles of chloroquine-treated chickens. The removal of chloroquine inhibition in vitro by the preparation of tissue homogenates resulted in increased proteolysis by lysosomal enzymes presumably due to an increase in available substrates derived from mitochondria and plasma membranes.

Glucose metabolism and dehydrogenase activities in the cytosol and mitochondria of mouse LS cells in chemostat culture

Energy metabolism has been examined in mouse LS cells growing under steady-state conditions in chemostat culture. The metabolic quotient of glucose oxidized (glucose consumed, but not fermented) remained constant, independent of growth rate between cell doubling times of 6 days and 1.2 days. Specific activities of cytochrome oxidase and malate dehydrogenase in the mitochondria remained constant at different growth rates, in accord with the constancy of the glucose oxidation rate. Cytosolic malate dehydrogenase activity was about fourfold greater than the mitochondrial isozyme. The steady-state rate of lactate production fluctuated because of technical limitations but correlated well with cytosolic lactate dehydrogenase activity.