Cell structure and the metabolism of insect flight muscle

The biochemical properties of insect flight muscle were investigated to ascertain the mechanisms whereby energy is made available for the contractile processes. It was found: 1. The endogenous respiration of muscle homogenates was diminished by starving the flies. The substrate for this respiration was probably glycogen. 2. To obtain the maximal rate of oxidation of glucose, the homogenate had to be fortified with inorganic phosphate, Mg ions, ATP, and cytochrome c. The nucleotides, AMP and ADP, were not as effective as ATP. The addition of DPN or TPN was not necessary for this system. 3. Flight muscle homogenates oxidized glycogen, some sugars, and amino acids, as well as the intermediates of the glycolytic and tricarboxylic acid cycles. Other evidence demonstrated the substrate specificity of the muscle. 4. By centrifugation, the muscle homogenate was divided into two fractions: one, a soluble fraction representing the sarcoplasm; the other, the particulate fraction which contained the fibrils and the sarcosomes. 5. The particulate fraction, alone, oxidized all the citric acid cycle intermediates, alpha-glycerophosphate, phosphopyruvate, and the amino acids, glutamic, proline, and cysteine. Regardless of the substrate, no oxygen uptake was found with the sarcoplasm by itself. 6. A recombination of the sarcoplasm and the particulate component was required for the oxidation of glycogen, the hexoses, and all the phosphorylated intermediates of glycolysis, except phosphopyruvate. 7. Isolated mitochondria accounted for all the enzymatic activity of the particulate fraction. These results demonstrate that the enzymes of intermediate metabolism are localized in the sarcoplasm or sarcosomes. The third cytological entity, the myofibrils, plays no role in the energy-providing scheme. From a functional viewpoint, the sarcoplasm and the mitochondria, in combination, furnish the energy for the actomyosin contraction. The results are discussed in relation to analogous findings in other insects and vertebrates.     

Cyclic AMP activation of respiration of the liver mitochondria in different metabolic states]

cAMP 10(-6) activates the liver mitochondria respiration in all the metabolic states and failed to change or increased the phosphorylation rate in the oxidation of saturating concentration of succinate and isocitrate. Preincubation of mitochondria or homogenate of the liver with cAMP is obligatory for this effect. The fraction V of serum albumin and EDTA did not prevent the effect. Noradrenaline enhanced the mitochondrial respiration only in incubation with the homogenate. The effect of noradrenaline and cAMP was not summed up. Probably the noradrenaline effect was mediated through cAMP. The data obtained are against the decisive role of the respiration and phosphorylation uncoupling or the oxidation substrate accumulation and lead to the assumption on the mitochondria enzymes activation.     

Oxidation of reduced glutathione by subcellular fractions of rat liver

1. A new method was used to diminish the autoxidation of GSH. 2. The oxidation of GSH by liver homogenates was studied with regard to concentration of homogenate, concentration of GSH, time, pH and anaerobiosis. 3. GSH was oxidized by recombinations of the supernatant with microsomes and with mitochondria. Each fraction alone caused little oxidation. 4. Proteins in the supernatant were required to obtain the effect, and low-molecular-weight compounds in the same fraction increased its effect. 5. GSH diminished the formation of malonaldehyde in homogenates. 6. GSH prevented a stimulating effect of the supernatant on the formation of malonaldehyde in microsomes and in mitochondria. 7. The malonaldehyde formation in microsomes together with the supernatant did not start until the concentration of endogenous low-molecular-weight thiols had decreased to a low level. 8. It is suggested that part of the oxidation of GSH in homogenates is coupled to a mechanism that counteracts the peroxidation of membrane lipids.     

Changes in the functional efficiency of flight muscle sarcosomes during heat death of adult Calliphora erythrocephala

The temperature sensitivity of flight muscle mitochondria from adult Calliphora erythrocephala has been studied. Intact flies were treated to sublethal and lethal high temperatures, their sarcosomes were isolated and the efficiency of the coupling of oxidation and phosphorylation was measured. The temperature sensitivity of sarcosomes is correlated with the heat death point of the intact animal and the impairment of oxidative phosphorylation with α-glycerophosphate suggests that a breakdown in ATP synthesis may be one of the causes of heat death. Restitution of enzyme activity was observed in mitochondria isolated from flies recovering from sublethal heat treatment. The results suggest that the impairment of membrane-enzyme complexes may be important lesions in heat death.     

Sodium benzoate inhibits fatty acid oxidation in rat liver: effect on ammonia levels

Sodium benzoate inhibited PC and octanoic acid-mediated State 3 respiration rates by 39 and 29%, respectively, at 0.5 mM in isolated rat liver mitochondria. At 2 mM, benzoate did not affect State 3 respiration rates with either succinate or malate plus glutamate, indicating that it did not act as an uncoupler. The oxidation of palmitate and octanoate was inhibited by 39 and 54% at 2 mM benzoate in liver homogenates. Benzoate, at 10 mmol/kg caused significant decreases in the levels of hepatic ATP, CoA, and acetyl-CoA. Administration of sodium benzoate to rats caused a dose-dependent increase in hepatic ammonia levels. However, the inhibitory effect of benzoate on fatty acid oxidation is not mediated through ammonia since ammonium chloride, at 1 mM, did not inhibit PC or octanoate oxidation in mitochondria or their oxidation in liver homogenate. Our results warrant a reevaluation of the use of sodium benzoate in the treatment of hyperammonemia.     

Changes in activity of some enzymes involved in glucose utilization and formation in developing rat liver

1. The activities of some enzymes involved in both the utilization of glucose (pyruvate kinase, ATP citrate lyase, NADP-specific malate dehydrogenase, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and NADP-specific isocitrate dehydrogenase, all present in the supernatant fraction of liver homogenates) and the formation of glucose by gluconeogenesis (glucose 6-phosphatase in the whole homogenate and fructose 1,6-diphosphatase, phosphopyruvate carboxylase, NAD-specific malate dehydrogenase and fumarase in the supernatant fraction) have been determined in rat liver around birth and in the postnatal period until the end of weaning. 2. The activities of those enzymes involved in the conversion of glucose into lipid are low during the neonatal period and increase with weaning. NADP-specific malate dehydrogenase first appears and develops at the beginning of the weaning period. 3. The marked increase in cytoplasmic phosphopyruvate carboxylase activity at birth is probably the major factor initiating gluconeogenesis at that time. 4. The results are discussed against the known changes in dietary supplies and the known metabolic patterns during the period of development.     

The characteristics of lactate oxidation in the tissue mitochondria of rats of different ages]

The parameters of respiration (V3, V4) and phosphorylation (the respiration control, ADP/O) have been studied using lactate as a substrate (obligatory with NAD addition) close by meaning to pyruvate on the liver and heart mitochondrion and homogenates of newborn rats. In 20-days and adult rats the mitochondria and homogenates oxidize the lactate (with NAD) with higher rate V4 but with lower value of respiration control as compared with the newborn animals. Simultaneously, a high activity of mitochondrial NADH-oxidase, oxidizing NADH, formed in the reaction of lactate dehydrogenase not connected with ATP synthesis. The role of mitochondrial NADH-oxidase are discussed as a factor increasing lactate oxidation, removing tissue lactate and activating the age dependent energy metabolism.     

The lipid droplet coat protein perilipin 5 also localizes to muscle mitochondria

Perilipin 5 (PLIN5/OXPAT) is a lipid droplet (LD) coat protein mainly present in tissues with a high fat-oxidative capacity, suggesting a role for PLIN5 in facilitating fatty acid oxidation. Here, we investigated the role of PLIN5 in fat oxidation in skeletal muscle. In human skeletal muscle, we observed that PLIN5 (but not PLIN2) protein content correlated tightly with OXPHOS content and in rat muscle PLIN5 content correlated with mitochondrial respiration rates on a lipid-derived substrate. This prompted us to examine PLIN5 protein expression in skeletal muscle mitochondria by means of immunogold electron microscopy and Western blots in isolated mitochondria. These data show that PLIN5, in contrast to PLIN2, not only localizes to LD but also to mitochondria, possibly facilitating fatty acid oxidation. Unilateral overexpression of PLIN5 in rat anterior tibialis muscle augmented myocellular fat storage without increasing mitochondrial density as indicated by the lack of change in protein content of five components of the OXPHOS system. Mitochondria isolated from PLIN5 overexpressing muscles did not possess increased fatty acid respiration. Interestingly though, (14)C-palmitate oxidation assays in muscle homogenates from PLIN5 overexpressing muscles revealed a 44.8% (P?=?0.05) increase in complete fatty acid oxidation. Thus, in mitochondrial isolations devoid of LD, PLIN5 does not augment fat oxidation, while in homogenates containing PLIN5-coated LD, fat oxidation is higher upon PLIN5 overexpression. The presence of PLIN5 in mitochondria helps to understand why PLIN5, in contrast to PLIN2, is of specific importance in fat oxidative tissues. Our data suggests involvement of PLIN5 in directing fatty acids from the LD to mitochondrial fatty acid oxidation.     

Tricarboxylic Acid Cycle Activity in Mitochondria from Soybean Nodules and Cotyledons

Infected cells of soybean (Glycine max) nodules require NADH,ATP, and 2-oxoglutarate for ammonia assimilation. The role ofmitochondria in nodule metabolism was investigated by determiningtheir respiratory properties and comparing them with cotyledonmitochondria. Nodule mitochondria oxidized malate at a ratetwice that of any other NAD-linked substrate although theirmalic enzyme activity was very low, accounting for only 12%of malate oxidation at pH 6.4 compared to 56% for cotyledonmitochondria. The reduction of NAD⁺ in mitochondria of noduleson adding malate (determined by fluorescence) was rapid andreached a stable level, whereas in cotyledon mitochondria theNADH level declined rapidly as oxaloacetate accumulated. Anoxaloacetate scavenging system in the mitochondrial reactionmedium increased malate oxidation by cotyledon mitochondria4-fold, but increased that of nodule mitochondria by less than50%. This demonstrates that the efflux of oxaloacetate by theoxaloacetate carrier is highly regulated by the extra-mitochondrialoxaloacetate concentration in cotyledon mitochondria comparedto nodule mitochondria. The activity of TCA cycle enzymes, exceptmalate and succinate dehydrogenases, was low in nodule mitochondria.Their oxaloacetate export during malate oxidation was rapid.The aspartate amino transferase activity associated with nodulemitochondria was sufficient to account for significant formationof 2-oxoglutarate from oxaloacetate and glutamate. These resultssuggest that nodule mitochondria operate a truncated form ofthe TCA cycle and primarily oxidize malate to provide oxaloacetateand ATP for NH₃ assimilation. Key words: Glycine max (L.), nitrogen fixation, gluconeogenesis, respiration     

Mitochondria from the hepatopancreas of the marine clam Mercenaria mercenaria: substrate preferences and salt and pH effects on the oxidation of palmitoyl-L-carnitine and succinate

A method is presented for the isolation of mitochondria with good respiratory control from the hepatopancreas of the marine clam Mercenaria mercenaria. Palmitoyl-L-carnitine is the preferred substrate of the mitochondria of the hepatopancreas based on state 3 rates of oxidation (in the presence of ADP). Rates of oxidation of pyruvate and glutamate were about one-half that of the lipid substrate in state 3. alpha-Glycerophosphate was oxidized at a rate about one-third that of palmitoyl-L-carnitine. All Krebs cycle intermediates were oxidized to some extent. Proline was not oxidized at detectable levels. The optimal range of KCl concentrations for the oxidation of palmitoyl-L-carnitine is between 250 and 500 mM whereas the optimal range of KCl concentration for the oxidation of succinate is between 200 and 350 mM. The optimal range of pH for the oxidation of succinate and for the oxidation of palmitoyl-L-carnitine lies between pH 6.5 and 7.5 based on the respiratory control ratio.     

Oxidative Activity of Particles prepared from Lettuce Seedlings

A particulate fraction similar to mitochondria was isolatedfrom germinating lettuce seedlings. The endogenous oxygen uptake of this fraction was very high.It could oxidize most of the Krebs cycle intermediates, theoxidation of l-malate being particularly marked. Glutamic, glycollic, and formic acids could also be oxidizedto a certain extent. Fatty acids inhibited the endogenous respiration,the inhibition increasing with increased concentration. Coumarinand thiouren do not seem to have any direct effect on the respiratoryenzyme systems. They apparently affect the respiration of wholeseeds only indirectly, maybe through some germination regulatingmechanism not yet known.     

ATP Accelerates Respiration of Mitochondria From Rat Lung and Suppresses Their Release of Hydrogen Peroxide

Lung mitochondria were isolated by differential centrifugation from pentobarbital-anesthetized male rats. One to three millimolar Mg²⁺-ATP increased the consumption of oxygen of lung mitochondria oxidizing 10 mM succinate > fourfold (P < 0.01) whereas ATP increased the respiration of liver mitochondria by < 35%. ATP also hyperpolarized partially uncoupled lung mitochondria in the presence of the mitochondria-specific antagonist, oligomycin. However, only 20% of the ATPase activity in the lung mitochondria was blocked by oligomycin compared to a blockade of 91% for liver mitochondria. We investigated the effect of reducing the non-mitochondrial ATPase activity in the lung preparation. A purer suspension of lung mitochondria from a Percoll gradient was inhibited 95% by oligomycin. The volume fraction identified as mitochondria by electron microscopy in this suspension (73.6± 3.5%) did not differ from that for liver mitochondria (69.1± 4.9%). ATP reduced the mean area of the mitochondrial profiles in this Percoll fraction by 15% (P <0.01) and increased its state 3 respiration with succinate as substrate by 1.5-fold (P < 0.01) with no change in the state 4 respiration measured after carboxyatractyloside. Hence, ATP increased the respiratory control ratio (state 3/state 4, P <0.01). In contrast, state 3 respiration with the complex 1-selective substrates, glutamate and malate, did not change with addition of ATP. The acceleration of respiration by ATP was accompanied by decreased production of H₂O₂. Thus ATP-dependent processes that increase respiration appear to improve lung mitochondrial function while minimizing the release of reactive oxygen species.     

Fertilization induced release of glycogen from bound state in sea urchin eggs

Glycogen in sea urchin eggs is found in both the precipitate and the supernatant fractions obtained by adding perchloric acid to the egg homogenate. Glycogen in the acid-insoluble fraction is apparently protein-bound (bound glycogen) while the acid-extractable form (free glycogen) seems to bind with less protein. The greatest amount of bound glycogen is found in the particulate fraction obtained by centrifugation of the egg homogenate at 10,000g for 30 minutes. The supernatant fraction obtained by centrifugation at 105,000g for two hours contained the largest amount of free glycogen of all the fractions obtained. The bound glycogen decreases and the free glycogen increases markedly following fertilization, while the total level of glycogen does not change. The glycogen release from the bound state occurs in vitro and the rate of release is higher in fertilized eggs than in unfertilized eggs. Polyamines (putrecine, spermidine, and spermine) cause an increase in the rate of glycogen release in the egg homogenate. cAMP, AMP, and ADP exert no effect on glycogen release in vitro, whereas ATP slightly enhances the rate of glycogen release. Na⁺ and K⁺ hardly accelerate the rate of glycogen release, and divalent cations, such as Ca²⁺ and Mg²⁺, cause an increase in the rate of glycogen release.     

Oxidative phosphorylation and respiratory control in mitochondria from Aspergillus niger

1. Tightly coupled mitochondria were isolated from Aspergillus niger by using an all-glass homogenizer followed by differential centrifugation. 2. The mitochondria oxidized the common intermediates of the tricarboxylic acid cycle, NADH(2) and the ascorbate-tetramethyl-p-phenylenediamine system. 3. High P/O ratios and control of respiration by ADP were obtained with all substrates tested. The average P/O ratios observed were: 1.5-1.8 with succinate as substrate [respiratory control ratio (RC) 2-4]; 0.8-1.0 with ascorbate-tetramethyl-p-phenylenediamine (RC 1.2-1.5); 1.4-1.8 with NADH(2) (RC 2-3); 2.4-2.8 with alpha-oxoglutarate (RC 3-5). 4. Bovine serum albumin (0.05-0.2%) was essential for tightly coupled respiration to be observed. 5. Coupled oxidation of exogenous NADH(2) was relatively insensitive to rotenone and Amytal. 6. The mitochondria responded to specific inhibitors and uncoupling agents in a manner similar to that of mammalian mitochondria. 7. It was concluded that the isolated mitochondria from A. niger show respiratory properties similar to those reported for intact yeast and mammalian mitochondria.     

Influence of glycogen storage on vascular smooth muscle metabolism

The role of glycogen as an oxidative substrate for vascular smooth muscle (VSM) remains controversial. To elucidate the importance of glycogen as an oxidative substrate and the influence of glycogen flux on VSM substrate selection, we systematically altered glycogen levels and measured metabolism of glucose, acetate, and glycogen. Hog carotid arteries with glycogen contents ranging from 1 to 11 micromol/g were isometrically contracted in physiological salt solution containing 5 mM [1-(13)C]glucose and 1 mM [1, 2-(13)C]acetate at 37 degrees C for 6 h. [1-(13)C]glucose, [1, 2-(13)C]acetate, and glycogen oxidation were simultaneously measured with the use of a (13)C-labeled isotopomer analysis of glutamate. Although oxidation of glycogen increased with the glycogen content of the tissue, glycogen oxidation contributed only approximately 10% of the substrate oxidized by VSM. Whereas [1-(13)C]glucose flux, [3-(13)C]lactate production from [1-(13)C]glucose, and [1, 2-(13)C]acetate oxidation were not regulated by glycogen content, [1-(13)C]glucose oxidation was significantly affected by the glycogen content of VSM. However, [1-(13)C]glucose remained the primary ( approximately 40-50%) contributor to substrate oxidation. Therefore, we conclude that glucose is the predominate substrate oxidized by VSM, and glycogen oxidation contributes minimally to substrate oxidation.     

Studies on guanine deaminase and its inhibitors in rat tissue

1. In kidney, but not in rat whole brain and liver, guanine-deaminase activity was localized almost exclusively in the 15000g supernatant fraction of iso-osmotic sucrose homogenates. However, as in brain and liver, the enzymic activity recovered in the supernatant was higher than that in the whole homogenate. The particulate fractions of kidney, especially the heavy mitochondria, brought about powerful inhibition of the supernatant guanine-deaminase activity. 2. In spleen, as in kidney, guanine-deaminase activity was localized in the 15000g supernatant fraction of iso-osmotic sucrose homogenates. However, the particulate fractions did not inhibit the activity of the supernatant. 3. Guanine-deaminase activity in rat brain was absent from the cerebellum and present only in the cerebral hemispheres. The inhibitor of guanine deaminase was located exclusively in the cerebellum, where it was associated with the particles sedimenting at 5000g from sucrose homogenates. 4. Homogenates of cerebral hemispheres, the separated cortex or the remaining portion of the hemispheres had significantly higher guanine-deaminase activity than homogenates of whole brain. The enzymic activity of the subcellular particulate fractions was nearly the same. 5. Guanine deaminase was purified from the 15000g supernatant of sucrose homogenates of whole brain. The enzyme separated as two distinct fractions, A and B, on DEAE-cellulose columns. 6. The guanine-deaminase activity of the light-mitochondrial fraction of whole brain was fully exposed and solubilized by treatment with Triton X-100, and partially purified. 7. Tested in the form of crude preparations, the inhibitor from kidney did not act on the brain and liver supernatant enzymes and the inhibitor from cerebellum did not act on kidney enzyme, but the inhibitor from liver acted on both brain and kidney enzyme. 8. The inhibitor of guanine deaminase was purified from the heavy mitochondria of whole brain and liver and the 5000g residue of cerebellum, isolated from iso-osmotic homogenates. The inhibitor appeared to be protein in nature and was heat-labile. The inhibition of the enzyme was non-competitive. 9. Kinetic, immunochemical and electrophoretic studies with the preparations purified from brain revealed that the enzyme from light mitochondria was distinct from enzyme B from the supernatant. A distinction between the two forms of supernatant enzyme was less certain. 10. Guanine deaminase isolated from light mitochondria of brain did not react with 8-azaguanine or with the inhibitor isolated from heavy mitochondria.     

Isolated mouse liver mitochondria are devoid of glucokinase

Glucokinase is a hexokinase isoform with low affinity for glucose that has previously been identified as a cytosolic enzyme. A recent report claims that glucokinase physically associates with liver mitochondria to form a multi-protein complex that may be physiologically important in apoptotic signaling [N.N. Danial, C.F. Gramm, L. Scorrano, C.Y. Zhang, S. Krauss, A.M. Ranger, S.R. Datta, M.E. Greenberg, L.J. Licklider, B.B. Lowell, S.P. Gygi, S.J. Korsmeyer, Nature 424 (2003) 952-956]. Here, we re-examined the association of glucokinase with isolated mouse liver mitochondria. When glucokinase activity was measured by coupled enzyme assay, robust activity was present in whole liver homogenates and their 9500 g supernatants (cytosol), but activity in the purified mitochondrial fraction was below detection (<0.2% of homogenate). Furthermore, addition of 45 mM glucose in the presence of ATP did not increase mitochondrial respiration, indicating the absence of ADP formation by glucokinase or any other hexokinase isoform. Immunoblots of liver homogenates and cytosol revealed strong glucokinase bands, but no immunoreactivity was detected in mitochondria. In conclusion, mouse liver mitochondria lack measurable glucokinase. Thus, functional linkage of glucokinase to mitochondrial metabolism and apoptotic signaling is unlikely to be mediated by the physical association of glucokinase with mitochondria.     

The control of tricarboxylate-cycle oxidations in blowfly flight muscle. The steady-state concentrations of citrate, isocitrate 2-oxoglutarate and malate in flight muscle and isolated mitochondria.

1. Blowfly (Phormia regina) flight-muscle mitochondria were allowed to oxidize pyruvate under a variety of experimental conditions, and determinations of the citrate, isocitrate, 2-oxoglutarate and malate contents of both the mitochondria and the incubation medium were made. For each intermediate a substantial portion of the total was present within the mitochondria. 2. Activation of respiration by either ADP or uncoupling agent resulted in a decreased content of citrate and isocitrate and an increased content of 2-oxoglutarate and malate when the substrate was pyruvate, APT and HCO3 minus. Such a decrease in citrate content was obscured when the substrate was pyruvate and proline owing to a large rise in the total content of tricarboxylate-cycle intermediates in the presence of proline and ADP. 3. An experiment involving oligomycin and uncoupling agent demonstrated that the ATP/ADP ratio is the main determinant of flux through the tricarboxylate cycle, with the redox state of nicotinamide nucleotide being of lesser importance. 4. Addition of ADP and Ca-2+ to activate the oxidation of both glycerol 3-phosphate and pyruvate, simulating conditions on initiation of flight, gave a decrease in citrate and isocitrate and an increase in 2-oxoglutarate and malate content. 5. There was a good correlation between these results with isolated flight-muscle mitochondria and the changes found in fly thoraces after 30s and 2 mihorax. 6. It is concluded that NAD-isocitrate dehydrogenase (EC 1.1.1.41) controls the rate of pyruvate oxidation in both resting fly flight muscle in vivo and isolated mitochondria in state 4 (nomenclature of Change & Williams, 1955).     

Metabolism of propionate by sheep liver: Pathway of propionate metabolism in aged homogenate and mitochondria

Experiments were conducted with aged nuclear-free homogenate of sheep liver and aged mitochondria in an attempt to measure both the extent of oxidation of propionate and the distribution of label from [2-¹⁴C]propionate in the products. With nuclear-free homogenate, propionate was 44% oxidized with the accumulation of succinate, fumarate, malate and some citrate. Recovery of ¹⁴C in these intermediates and respiratory carbon dioxide was only 33%, but additional label was detected in endogenous glutamate and aspartate. With washed mitochondria 30% oxidation of metabolized propionate occurred, and proportionately more citrate and malate accumulated. Recovery of ¹⁴C in dicarboxylic acids, citrate, α-oxoglutarate, glutamate, aspartate and respiratory carbon dioxide was 91%. The specific activities of the products and the distribution of label in the carbon atoms of the dicarboxylic acids were consistent with the operation solely of the methylmalonate pathway together with limited oxidation of the succinate formed by the tricarboxylic acid cycle via pyruvate. In a final experiment with mitochondria the label consumed from [2-¹⁴C]propionate was entirely recovered in the intermediates of the tricarboxylic acid cycle, glutamate, aspartate, methylmalonate and respiratory carbon dioxide.     

Fatty acid oxidation in human and rat heart. Comparison of cell-free and cellular systems

Oxidation rates of palmitate and activities of the mitochondrial marker enzymes cytochrome c oxidase and citrate synthase have been determined in homogenates, isolated mitochondria and slices of human and rat heart and in calcium-tolerant rat cardiac myocytes. Homogenates and mitochondria from rat heart showed a 6- and 2.5-fold higher palmitate oxidation rate than the corresponding preparations from human heart. From the palmitate oxidation rates and cytochrome c oxidase and citrate synthase activities as parameters, the mitochondrial protein contents of human and rat heart were calculated to be about 18 and 45 mg/g wet weight, respectively. Based on citrate synthase activities, the fatty acid oxidation rates were about the same in homogenates and isolated mitochondria, much lower in myocytes and lowest in slices. In the cellular systems the palmitate molecule was more completely oxidized than in homogenates or isolated mitochondria. Fatty acid oxidation rates were concentration-dependent in slices, but not with myocytes. With the cellular systems, palmitate oxidation was synergistically stimulated by the addition of carnitine, coenzyme A and ATP to the incubation medium. This stimulation could be attributed only partly to an increased oxidation in damaged cells.