Rate Of Succinate Oxidation Research Articles

The equilibrium between the mitochondrial ATPase (F 1) and its natural inhibitor in submitochondrial particles

1. The reversible equilibrium between the mitochondrial ATPase (F 1) and its naturally occurring inhibitor in Mg-ATP submitochondrial particles has been studied under different conditions. 2. High ionic strength favours dissociation of the ATPase inhibitor as tested by ATPase and ATP-driven transhydrogenase activities. 3. Dissociation of the ATPase inhibitor results in an increased maximal velocity of the ATPase activity measured in the presence of uncoupler and an increased affinity for adenine nucleotides, in particular for ATP. 4. Association of the ATPase inhibitor with inhibitor-depleted Mg-ATP particles causes a slowing of the initial rate of succinate oxidation. 5. The antibiotic aurovertin stimulates the ATPase activity of Mg-ATP particles preinculbated in the presence of a supply of oxidative energy. Bound aurovertin impedes the association of inhibitor-deficient particles with ATPase inhibitor. 6. The fluorescence of aurovertin bound to inhibitor-containing particles is much less than that of aurovertin bound to inhibitor-depleted particles. 7. The oligomycin-sensitivity-conferring protein, added either alone or in the presence or absence of membranous components of the ATPase complex, has little or no effect on the fluorescence of the F 1-aurovertin complex. 8. It is suggested that the ATPase inhibitor brings F 1 in a conformation denoted ∗F 1 that binds aurovertin with a low quantum yield, a decreased affinity and an increased binding capacity.

Low-temperature spectral properties of the respiratory chain cytochromes of mitochondria from Crithidia fasciculata

Highly purified mitochondrial preparations from the trypanosomatid hemoflagellate, Crithidia fasciculata (A.T.C.C. No.11745), were examined by low-temperature difference spectroscopy. The cytochrome a+ a 3 α maximum of hypotonically-treated mitochondria reduced with succinate, was shifted from 605 nm at room temperature to 601 nm at 77 °K. The Soret maximum, found at 445 nm at 23 °C, was split at 77 °K into two approximately equally absorbing species with maxima at 438 and 444 nm. A prominent shoulder observed at 590 nm with hypotonically-treated mitochondria was not present in spectra of isotonic controls. The cytochrome b maxima observed in the presence of succinate plus antimycin A were shifted from the 431 and 561 nm positions observed at 23 °C to 427 and 557 nm at 77 °K. Multiple b cytochromes were not apparent. Unlike other soluble c-type cytochromes, the α maximum of cytochrome c 555 was not shifted at 77 °K although it was split to give a 551 nm shoulder adjacent to the 555 nm maximum. This lack of a low-temperature blue shift was true for partially purified hemoprotein preparations as well as in situ in the mitochondrial membrane. Using cytochrome c 555-depleted mitochondria, a cytochrome c 1 pigment was observed with a maximum at 420 nm and multiple maxima at 551, 556, and 560 nm. After extraction of non-covalently bound heme, the pyridine hemochromogen difference spectrum of cytochrome c 555-depleted preparations exhibited an α maximum at 553 nm at room temperature. The reduced rate of succinate oxidation by cytochrome c 555-depleted mitochondria and the ferricyanide requirement for the reoxidation of cytochrome c 1, even in the presence of antimycin, indicated that cytochrome c 555-mediated electron transfer between cytochromes c 1 and a+ a 3 in a manner analagous to that of cytochrome c in mammalian mitochondria.

Effect of thyroid hormone on respiratory control of liver mitochondria from adult and senescent rats

Changes in the State-3 (ADP-stimulated) and State-4 (controlled) rates of succinate oxidation in liver mitochondria induced by thyroid hormone were examined in adult (12–13 months old) and senescent (27–29 months old) rats. Adult animals, made hypothyroid by feeding thiouracil, had significantly decreased State-3 rates of respiration and respiratory control ratios. The State-4 rate of oxidation was not lowered significantly. A single injection of triiodo- l-thyronine (T 3) to these hypothyroid rats resulted in increases in States-3 and -4 rates of respiration as well as in the respiratory control ratios. The enhancement of the State-3 rate was evident at 6 hr after hormonal administration, reaching a maximum (2.3-fold the hypothyroid value) in about 2 days, with this high level being maintained for at least an additional 3 days. The increase in State-4 rate of succinate oxidation was 1.6-fold; this was attained in 1 day after injection of T 3 and was maintained for at least an additional 4 days. The rates of induction by T 3 of the increases in States-3 and -4 rates of succinate oxidation and respiratory control ratio as well as the maximal levels achieved with hormonal administration were the same in adult and senescent rats. These findings suggest that the biochemical machinery necessary for the stimulation in respiratory rates and respiratory control ratio in the livers of aged animals is in no way impaired and that senescent rats are fully capable of responding, at least in this system, to the direct adaptive stress of thyroid hormone.

Some Reactions of Isolated Corn Mitochondria Influenced by Juglone

The effects of juglone on the uptake of O2 by excised corn roots (Zea mays L., Wf9 cms- T × M14) and isolated corn mitochondria arc reported. The O2 uptake by excised corn roots, as measured by an O2 electrode, was inhibited more than 90% after a one-hour treatment of 500 μM juglone. Lesser inhibitions were observed with 50 μM and 250 μM juglone. In a KC1 reaction medium in the absence of inorganic phosphate (Pi), juglone stimulated the rate of O2 uptake by isolated mitochondria oxidizing NADH, succinate, or malate + pyruvate. In the presence of Pi, juglone concentrations of 3 μM and greater inhibited the state 3 oxidation rates of succinate and malate + pyruvate, lowered respiratory control and ADP/O ratios obtained from the oxidation of NADH, malate + pyruvate, or succinate, and reduced the coupled deposition of calcium phosphate within isolated mitochondria driven, by the oxidation of malate + pyruvate. The inhibition of state 3 O2 uptake by isolated mitochondria, an oxidative state in which electron transfer is coupled to ATP production, is seen to correlate with the inhibition affected by juglone when applied to tissues in vivo.

Functional and structural changes in liver mitochondria of rats due to CCl 4 intoxication—I : Studies on state of electron-transport chain

Functional changes in the electron-transport chain in the liver mitochondria of rats, induced by carbon tetrachloride treatment, have been studied in this work. Experimental evidence led us to the conclusion that along with uncoupling of oxidative phosphorylation, impairment of the respiratory chain is observed. The decrease in the rate of succinate oxidation is thought to be due to a deficit in cytochrome c. The marked respiratory inhibition observed during NAD-linked substrate oxidation is considered to be caused by impairment of either the first complex or the substrate dehydrogenases.

Effect of pH on the oxidation of malate by isolated cauliflower bud mitochondria

The effects of pH on the rate and products of malate oxidation by isolated cauliflower bud mitochondria are described. Between pH 6·0 and 7·0 pyruvate is the major product of the oxidation and the rate of oxidation increases with increasing pH. Similar increases in α-oxoglutarate and succinate oxidation rates occur and a general effect of pH on mitochondrial electron transport is indicated. Between pH 7·0 and 8·0 a switch in the major product of malate oxidation from pyruvate to oxaloacetate occurs and the oxidation becomes subject to a strong product inhibition. This change over in products is interpreted in terms of the pH-activity profiles of mitochondrial malate dehydrogenase and NAD requiring malic enzyme.

Diurnal variations in enzymic activities in subcellular fractions of cactus phylloclades

The activities of phosphopyruvate carboxylase (E.C. 4.1.1.31), malic enzyme (malate dehydrogenase (decarboxylating) E.C. 1.1.1.40), selected enzymes of TCA cycle and electron transport chain were determined in mitochondrial and supernatant fractions isolated from phylloclades of the cactus Nopalea dejecta, collected at noon and midnight. There were significant and consistent increases in the activities of mitochondrial and supernatant phosphopyruvate carboxylase, mitochondrial aconitate hydratase (E.C. 4.2.1.3) and soluble malate dehydrogenase (E.C. 1.1.1.37) and decreases in mitochondrial malate dehydrogenase and fumarate hydratase (E.C. 4.2.1.2) and in both mitochondrial and soluble malic enzyme in the midnight samples. Of the respiratory chain enzyme complexes tested, cytochrome c oxidase (E.C. 1.9.3.1) and succinate cytochrome c reductase had higher activities at noon, while reduced NAD-cytochrome c reductase and reduced NAD dehydrogenase (E.C. 1.6.99.3) activities were higher at midnight. The rate of succinate oxidation and coupled phosphorylation also varied, with a maximum at noon. The diurnal pattern of alteration in enzymic activities has a bearing on crassulacean acid fluctuation.

Effects of staphylococcus alpha-toxin and streptolysin S on the oxidation of succinate by ascites tumour cells.

Summary Small amounts of purified staphylococcus α-toxin caused an immediate increase in the rate of succinate oxidation by Krebs 2 mouse ascites tumour cells. This effect appeared to be specific for α-toxin and not to be due to contamination of the α-toxin preparation with staphylococcus δ-toxin. During interaction with Krebs 2 cells, the haemolytic activity of α-toxin decreased by more than 95 per cent. Streptolysin-S preparations also caused increased oxidation of succinate, but only when washed cells were pretreated with toxin. These effects can be explained by postulating an action of the toxins on the cell membrane that leads to increased permeability to succinate. The different actions of staphylococcus α-toxin and streptolysin S are discussed.

Electron transport in beef heart muscle preparation The inhibition of ubiquinone-stimulated succinate oxidase activity by antimycin and a quinoline-N-oxide derivative

1. The succinate oxidase activity of acetone extracted heart muscle preparation could be restored by the addition of ubiquinone homologues.2. Where short chain ubiquinone homologues were employed to restore succinate oxidase activity, the reconstituted system was found to be no longer fully inhibited by antimycin.3. When the acetone extracted preparation was preincubated with ubiquinone homologues and then washed free of unbound quinone, the succinate oxidase of the reconstituted particle was found to be completely inhibited by antimycin.4. Previous reports had proposed that the quinone mediated antimycin insensitive electron was due either to an electronic by-pass around the antimycin sensitive site or to the displacement of inhibitor by the added quinone.These two alternatives were investigated by measuring the sensitivity of acetone extracted heart muscle preparation at different concentrations of inhibitors in the presence of quinone.5. The results appear to support an electron by-pass mechanism of electron flow mediated by hydrophilic quinones Q-o and methyl-Q-o. The pattern of the reversal of antimycin inhibition by the higher ubiquinone homologues Q-2 and Q-3 suggests that displacement of inhibitor is the controlling factor.6. The Q-o and methyl-Q-o mediated antimycin insensitive rate of succinate oxidation of both extracted and unextracted heart muscle was compared and results suggest that this pathway of electron flow does not involve the endogenous ubiquinone of the respiratory chain.7. These findings are further considered in relation to the hydrophilic nature of short chain ubiquinone homologues.

Effects of cold exposure and hibernation on the liver, brown fat, and testes from golden hamsters.

AbstractAdult, male hamsters, (Mesocricetus auratus, Waterhouse), were exposed to an environment of 5 C (2 months and 3 to 4 months) to investigate the effects of cold‐exposure and 2 to 5 days of hibernation on the following parameters: (1) body weights, (2) testes weights, (3) testes and brown fat histology and histochemistry, and (4) liver and brown fat mitochondrial succinate oxidation. Controls were maintained at room temperature. Only the hibernating animals lost a significant amount of weight during exposure. Although the testes regressed in both cold‐exposed and hibernating animals, the testes of hibernators showed some signs of recrudescence relative to the cold‐exposed non‐hibernators, as indicated by increased spermatogenic activity, slightly larger seminiferous tubules, greater organ weights, and the presence of PAS positive, diastase sensitive material. Brown fat in experimental animals had smaller and more numerous fat droplets per cell than the controls and nuclei which appeared to be more centrally located. Analysis of liver mitochondrial activity revealed the rate of succinate oxidation to be reduced in hibernators and increased in cold‐exposed non‐hibernators. Brown fat succinate oxidation was unaltered in both experimental groups. These results indicate that, early in the hibernation cycle, both physiological and morphological changes occur which distinguish hibernating hamsters from cold‐exposed hamsters which failed to hibernate.

Respiration and mitochondrial content in single neurons of the supraoptic nucleus. A correlative study in osmotic stress.

The study was undertaken to investigate the possible correlation of total volume of mitochondria per cell with the rate of succinate oxidation in isolated nerve cell bodies, after various functional stresses in the experimental animals. Significant cytological effects were found in the nerve cells of the supraoptic nucleus in rats which had been thirsting for 4-12 days or had been given 2% sodium chloride solution as a substitute for drinking water for a few weeks. Quantitation of mitochondria was done from electron micrographs. The cell volumes were calculated from sections of Epon-embedded tissue under phase-contrast microscopy. Succinate oxidation was measured on groups of 10 nerve cells with the microdiver technique. As a result of either thirst or sodium chloride load, the volume of mitochondria per nerve cell more than doubled. The rate of succinate oxidation was not changed after the rats had been thirsting but was enhanced by over 100% after they had drunk sodium chloride. A linear relationship was found for the amount of mitochondria versus respiration in the supraoptic neurons for all experimental groups except the thirsting animals. The mitochondria in the supraoptic neurons from thirsting animals were of the same size or smaller than those in controls, whereas in animals given sodium chloride solution the mitochondria were considerably enlarged. The observed effects were specific for the supraoptic nucleus.

Integrity and Respiration of Red Beet Mitochondria Isolated and Examined in Different Sucrose Concentrations

AbstractRespiration experiments with succinate as substrate were made with red beet mitochondria isolated in soluitions containing 0.25 to 1.25 M sucrose. The respiration was measured in reaction media adjusted to be 0.25, 0.50, 0.75 or 1.0 osmolar.With mitochondria isolated in 0.25 or 0.50 M sucrose the rate of succinate oxidation was completely dependent on the osmotic pressure of the reaction medium (decreasing with increasing osmotic pressures). Isolation in 0.75 M sucrose caused a slight after‐effect of the osmotic pressure of the isolation medium, and by isolation in 1.0 M or 1.25, M sucrose the after‐effect was complete. The rate of oxidation was low and independent of the osmotic pressure of the reaction medium.An electron microscopic examination of the state of the mitochondria before and after the respiration period showed that with the conditions used in the present experiments the structure of the mitochondria remained well preserved regardless of the osmotic pressures used.

Properties of Higher Plant Mitochondria. III. Effects of Respiratory Inhibitors

The effects of representative respiratory inhibitors were investigated on the coupled respiration of mung bean mitochondria using succinate and l-malate as substrates. The inhibitors studied were: (I) malonate, (II) amytal and rotenone, (III) antimycin A and 2-n-nonyl-4-hydroxyquinoline N-oxide (NOQNO), and (IV) cyanide and azide.Malonate inhibition of succinate oxidation follows a classical type of competitive inhibition with an inhibitor dissociation constant of 0.13 mm. There is no inhibition detectable when malate is used as substrate. In contrast to animal mitochondria, amytal is capable of inhibiting 20 to 40% of succinate oxidation and 90 to 100% of malate oxidation, but inhibition due to rotenone amounts to only 0 to 20% of succinate oxidation and 40 to 50% of malate oxidation. The half-maximal inhibition caused by amytal occurs at 2 to 2.5 mm and that by rotenone at 3 mmumoles/mg protein.The maximal inhibition caused by either antimycin A or NOQNO is 70 to 80% of the state 3 respiration. Very little inhibition was observed on the state 4 respiration, and both inhibitors were capable of titrating stoichiometrically with mitochondrial protein with identical titers, 0.22 mmumoles/mg protein for half-maximal inhibition. They differ, however, in that NOQNO does uncouple oxidative phosphorylation in mung bean mitochondria, but antimycin A does not do so. Both cyanide and azide inhibit the state 3 rate 65 to 80%. Inhibition of state 4 respiration can be up to 50% by cyanide, while almost none by azide. Uncoupling action was noted with cyanide, but very little with azide.It is concluded that the second state 3 rate of succinate oxidation includes 80% succinoxidase, the remaining 20% being contributed by the NADH pathway. Malate oxidation apparently does not involve succinoxidase. Malate oxidation is completely sensitive to amytal, but only 50% inhibited by rotenone. A difference between animal and plant mitochondria appears to be in the flavoproteins associated with NADH oxidation.From the observations that antimycin A, NOQNO, cyanide, and azide do not cause complete inhibition, it is suggested that a leakage of electrons to oxygen exists before the site of inhibition of antimycin A or NOQNO.

Comparative activity and intracellular distribution of tricarboxylic acid cycle enzymes in Haemonchus contortus larvae and rat liver

1. 1. The enzymes involved in the tricarboxylic acid cycle were investigated in Haemonchus contortus and the results compared with those obtained for the corresponding enzymes in rat liver. 2. 2. Succinic dehydrogenase and the oxoglutarate dehydrogenase system were detected in a particulate preparation from H. contortus, their specific activities being about one-third those of the corresponding enzymes in rat liver. The succinic dehydrogenase of H. contortus resembled that of facultative anaerobes in its relative rates of succinate oxidation and fumarate reduction. 3. 3. The level of citrate synthase activity in H. contortus crude homogenates was much greater than that in rat liver, the bulk of this activity being retained by the supernatant fraction. In rat liver the specific activities of the supernantant and particulate fractions were almost equal. 4. 4. The level of NADP specific isocitrate dehydrogenase in H. contortus was about one-sixth that in rat liver, although the intracellular distribution of this enzyme was similar in both tissues, the supernatant containing most of the activity. 5. 5. In contrast to the exclusive localization of NAD specific isocitrate dehydrogenase in the mitochondrial fraction of rat liver homogenates, considerable activity was detected in the supernatant fraction of H. contortus homogenates as well as in the particulate fraction. Like that of vertebrate tissue, the NAD specific isocitrate dehydrogenase of H. contortus was activated by ADP. 6. 6. Aconitate hydratase, fumarate hydratase and malate dehydrogenase were present in H. contortus and their intracellular distribution and specific activities were very similar to those of the corresponding rat liver enzymes. 7. 7. The particulate fraction from H. contortus was found to be a poor source of some of the enzymes of the tricarboxylic acid cycle, especially of aconitate hydratase and NADP specific isocitrate dehydrogenase, much higher levels of activity being retained by the supernatant. Similar results were obtained with rat liver homogenates and the implications of these findings in relation to previous reports on the existence of this cycle in other parasites is discussed. 8. 8. It appears that a full tricarboxylic acid cycle can operate in H. contortus under aerobic conditions and the possible role of these enzymes under anaerobic conditions is discussed.

Succinate oxidation by human liver mitochondria

Factors controlling the rate of oxidation of succinate were studied in mitochondria isolated from twenty-seven specimens of normal human liver obtained at operation. These were compared with mitochondria prepared from the livers of anesthetized and unanesthetized rats. Previously reported lower rates of succinate oxidation by human liver mitochondria were confirmed. This difference was no longer apparent when the succinic dehydrogenase activities of mitochondrial acetone powders were compared. The apparent K m of succinate for human liver mitochondrial succinoxidase is less than that of the anesthetized and unanesthetized rat. The concentration of oxalacetate is greater in human liver mitochondria compared with the anesthetized and unanesthetized rat. In both aerobic- and cyanide-inhibited mitochondria, the extent of the apparent succinate linked reduction of mitochondrial pyridine nucleotide was less in man than in the rat. In aerobic rat liver mitochondria, anesthesia lowers the extent of succinate-linked pyridine nucleotide reduction.

The influence of thyroid hormones on normal rat-liver mitochondria II. Thyroxine effects on pretreated mitochondria

1. 1. Dilution of a sucrose suspension of mitochondria with an equal volume of deionized H 2O lowered the respiratory control and permitted a number of thyroxine- induced effects to be observed. 2. 2. The most immediate effect of thyroxine was an increase in the rate of succinate oxidation. This was followed at about 1 min by a further drop in respiratory control and at 2 min by an increase in adenosine-5′-triphosphatase activity. These effects all contributed to a reduction in phosphorylation efficiency in the presence of thyroxine. 3. 3. Although thyroxine increased the rate of mitochondrial swelling, beginning at approx. 2 min after the start of the incubation, this effect was clearly not associated with any of the other thyroxine effects except possibly the stimulation of adenosine-5′-triphosphatase activity. 4. 4. The results support other work in which thyroxine stimulates succinate oxidation under conditions in which it is not limited by the phosphorylative capacity of the mitochondria.

Spectrophotometric and kinetic studies of the reconstituted succinate oxidase system

Summary Some spectral and kinetic properties of the reconstituted succinate oxidase system of Keilin and King have been studied. 1. With the reconstituted enzyme system the total rate of succinate oxidation was not affected by dilution. 2. The distinct absorption peaks that appeared at 603, 562, 552 and 442 m μ , are essentially the same as those observed for the untreated heart-muscle preparation except for a slight shift of the α -peak of cytochrome a from 604 to 603 m μ . 3. A partial inactivation and/or destruction of the cytochromes may have occurred during the alkali treatment of heart-muscle preparation as indicated by the decrease in the extent of enzymic reduction by succinate as well as their low turnover number. 4. The velocity constants for the sequential reactions of succinate oxidase have been calculated. The magnitudes of these constants are quite similar to those obtained for the untreated heart-muscle preparation. 5. Kinetic evidence indicates that the soluble fraction derived from the alkali treatment of the heart-muscle preparation has a competitive inhibitory effect in relation to the active soluble succinate dehydrogenase (succinate:(acceptor) oxido-reductase, EC 1.3.99.1) on the reduction of cytochrome b by succinate. 6. The molecular relationship between succinate dehydrogenase and cytochrome b as well as the participation of cross-linked respiratory chain assemblies during succinate oxidation are discussed.

NOCTURNAL CHANGES IN OXIDATIVE ACTIVITIES OF RAT LIVER MITOCHONDRIA.

Male rats kept on an artificial day-night cycle were active at night and rested during the day. The rate of succinate oxidation in liver mitochondria from these animals was 40 percent greater at night. Significant nocturnal increases in the ratio of phosphate to oxygen were obtained with citrate, alpha-ketoglutarate, and malate.

The succinic oxidase and DPNH oxidase systems of the bay mussel, Mytilus edulis

Respiratory particles have been prepared from adductor muscles of four lamellibranch mollusks. Nearly a tenfold increase in specific activity for succinic oxidase has been obtained. The respiratory particles of posterior adductor muscles of bay mussel, Mytilus edulis, have been studied in more detail. They can catalyze the oxidation of succinate, malate and reduced diphosphopyridine nucleotide (DPNH) by molecular oxygen and by mammalian cytochrome c. The rate of succinate oxidation by oxygen is stimulated by exogenous cytochrome c and inhibited by antimycin A or cyanide. The rate of DPNH oxidation by oxygen is slightly inhibited by the addition of amytal, antimycin, or cyanide and greatly increased by cytochrome c. In the presence of cytochrome c, DPNH oxidation is potently inhibited by cyanide. The existence of cytochrome oxidase in mussel particles is demonstrated by their capability to oxidize ascorbate and reduced cytochrome c. The convergence of the succinate and the DPNH respiratory chains is demonstrated from the simultaneous oxidations of these substrates; rates of these oxidations are not additive. The existence of absorption bands similar to cytochromes c-c 1, b, and a-a 3 of sarcosomal components has been shown by direct spectroscopic examination of mussel particles.

Studies on isolated human skeletal muscle mitochondria

Mitochondria prepared from human skeletal muscle tissue have been analyzed for oxidation rates with different substrates, P O ratios, ATPase activities, P i-ATP exchange rates and respiratory control ratios. Human skeletal muscle mitochondria oxidize at high rates pyruvate (+malate), α-ketoglutarate and glutamate, and at lower rates all other Krebs cycle metabolites, glycerol-1-phosphate and fatty acids. The oxidation rate of succinate is increased several times if oxaloacetate is removed from the medium by the addition of cysteine sulphinic acid, or if its formation is prevented by the addition of amytal. Adequate P O ratios are obtained with most substrates tested. Addition of 2,4-dinitrophenol (DNP) causes an inhibition of the oxidation of glutamate, but not of α-ketoglutarate, and the inhibition goes parallel to the uncoupling effect. Addition of adenine nucleotides partially protects against the inhibition of glutamate oxidation. Complete abolishment of respiratory control is obtained with 10 −5 M DNP; this concentration of DNP lowers the P O ratio only to a small extent, and induces only a slight ATPase activity. Serum albumin can partially protect against the uncoupling induced by DNP and other agents. Human skeletal muscle mitochondria show an ATPase activity which is stimulated by low concentrations and inhibited by high concentrations of Mg 2+. 10 −4 M DNP induces a high ATPase activity, which is inhibited by high Mg 2+ concentrations. The respiratory control ratio of human skeletal muscle mitochondria with pyruvate (+malate) is about 5–6 in the presence, and 10 or more in the absence of added ATP. The respiratory control ratio obtained in the presence of ATP is inversely related to the level of the Mg 2+-stimulated ATPase activity, whereas no such relation is found in the absence of added ATP. Also no relation is found between the level of Mg 2+-stimulated ATPase activity and the P O ratio with pyruvate (+ malate) or succinate. The hypothesis is advanced that the Mg 2+-stimulated ATPase of fresh skeletal muscle mitochondria is different in mechanism from the Mg 2+-stimulated ATPase found in aged liver mitochondria. The results are discussed in regard to the mechanism of mitochondrial respiratory control and its relation to oxidative phosphorylation and ATPase activities.