Effect of filipin on Rat-liver and yeast mitochondria

1. Under the appropriate conditions intact yeast and mammalian mitochondria exhibit a heretofore unobserved sensitivity to the polyene antibiotic, filipin. The activity of the “filipin complex” (Filipins I, II, III and IV) is shown to be primarily due to the component designated Filipin II.

2. Yeast mitochondria treated with filipin complex, or purified Filipin II, exhibit “uncoupled” succinate oxidation and inhibited -ketoglutarate oxidation. Maximum filipin effect is observed at a concentration of 4 mM Filipin II. Rat-liver mitochondria are more sensitive to filipin than yeast mitochondria, and respiratory inhibition is observed regardless of substrate.

3. In liver mitochondria filipin-inhibited respiration is not relieved by Mg²⁺, K⁺, Ca²⁺ or 2,4-dinitrophenol, but is reversed by cytochrome c.

4. It is proposed that filipin treatment leads to altered membrane permeability and that respiratory inhibition is due to a loss of endogenous respiratory cofactors or an inactivation of primary dehydrogenases. The filipin-uncoupled yeast respiration may likewise be attributed to an altered phosphate permeability of the yeast mitochondrial membranes.     

Studies on yeast mitochondria: Some properties of mitochondria isolated from Saccharomyces Carlsbergensis grown in normal glucose medium

1. Mitochondria isolated from Saccharomyces Carlsbergensis are found to have three phosphorylation sites in the respiratory chain for the oxidation of NADH and NAD⁺-linked substrates and two for succinate oxidation. Freshly isolated mitochondria exist in an inhibited state with no respiratory control, but on ageing for 2–3 h a good coupled state is obtained. -Ketogultarate and -glycerophosphate are poorly oxidized in these mitochondria.

2. Exogenous NADH is a very good substrate for yeast mitochondrial respiration and apparently has a very low K_m. However, one-third of the added NADH is not available for oxidation probably due to some form of compartmentation. Studies of both oxygen uptake and the redox changes of cytochrome b show complete oxidation of two-third of the added NADH.

3. Difference spectra of yeast mitochondria at liquid-nitrogen temperatures show all the characteristic peaks of cytochromes a (600 nm), b (558, 525 and 428 nm), c₁ (552 nm) and c (545 and 516 nm).

4. The reduction of cytochrome b by dicumarol in antimycin A inhibited mitochondria provides evidence for an energy conservation site on the substrate side of cytochrome b.

5. In the absence of added ADP, the oxidation of malate and pyruvate occurs in the yeast mitochondria in a new respiratory state (State X) where the oxygen uptake occurs at State 4 rate but the redox level of the flavins, cytochrome b and c are similar to State 3. State X respiration is believed to be due to depletion of the high energy intermediate C I caused by the substrate anions accumulation.

6. The responses of yeast mitochondria to Ca²⁺ are qualitatively similar to those in rat liver mitochondria, particularly with respect to respiratory stimulation, membrane alkalinization and its accumulation in the mitochondria with succinate as the substrate in the presence and absence of acetate.     

Mitochondrial oxygen affinity as a function of redox and phosphate potenitals

1. The conditions under which mitochondria might catalyse a net reversal of oxidative phosphorylation are analysed.

2. Rat-liver mitochondria, incubated under such conditions, show a strongly diminished affinity for oxygen.

3. The velocity of respiration under these conditions is a hyperbolic function of the oxygen concentration.

4. The K_m for oxygen is less than 0.1 μM at low phosphate potential, irrespective of substrate, and 1–3 μM under reversal conditions.

5. The observed kinetics can be accounted for in a simple mechanism for cytochrome oxidase action.     

Oxidative phosphorylation in nuclei isolated from rat thymus

1. Nuclei were isolated from rat thymus in 0.25 M sucrose-3 mM CaCl₂. Fractionation of thymus tissue showed that the nuclear fraction could be contaminated with 10% intact cells at the most.

2. The nuclear fraction contained about 50% of the cellular ATP. Under anaerobic conditions nuclear ATP is degraded. When, after an anaerobic incubation, the nuclei are incubated under aerobic conditions endogenous nuclear ATP is resynthesized. The amount of ATP found cannot be accounted for by mitochondria or intact cells. It could also be shown that the ATP synthesis is not due to such contamination.

3. Nuclear ATP synthesis and respiration were inhibited by amytal, rotenone, and carbon monoxide. The latter inhibition was reversed by light. 2,4-Dinitrophenol abolished ATP synthesis and accelerated respiration. Oligomycin inhibited respiration and phosphorylation. The inhibition of respiration by oligomycin could be released by dinitrophenol. ATP synthesis was also inhibited by lewisite. This inhibition could be reversed by BAL. No inhibition, however, was found with arsenite or arsenite + BAL.

4. A P:O ratio between 0.6 and 1.0 was obtained for nuclear phosphorylation. This must be considered as a minimal value.

5. It is concluded that thymus nuclei are capable of respiratory-chain-linked oxidative phosphorylation.     

Measurement of steady-state values of respiration rate and oxidation levels of respiratory pigments at low oxygen tensions. A new technique

1. An apparatus was developed for the simultaneous measurement of steady-state values of respiration rate and oxidation level of respiratory pigments at low oxygen tensions. An open reaction system is utilized. The liquid sample is in contact with a gas mixture whose oxygen tension can be increased linearly with time at a rate so slow that the system is always practically at a steady state.

2. Assuming Michaelis-Menten kinetics in the respiration, theoretical curves for oxygen tension in the liquid and oxidation level of the terminal oxidase during a linear increase of the oxygen tension in the gas were calculated.

3. Measurements were performed on rat liver mitochondria. Steady-state curves for oxygen tension in the liquid and oxidation level of the terminal oxidase, cytochrome a₃, obtained with coupled mitochondria resembled the theoretical curves. For uncoupled mitochondria the cytochrome a₃ curve was signmoidal, deviating strongly from the theoretical curve.

4. The apparent K_m for oxygen uptake of coupled mitochondria in the presence of pyruvate and malate, in the absence of phosphate was found to be 0.5 μM. In the case of uncoupled mitochondria the oxygen tension in the liquid could not be measured with sufficient accuracy to allow comparison with Michaelis-Menten kinetics. The apparent K_m for oxygen uptake was less than 0.05 μM.     

Bongkrekic acid sensitivity of respiration-deficient mutants and of petite-negative species of yeasts

1. Growth on glucose of cytoplasmic respiration-deficient (ρ⁻) mutants isolated from five strains of Saccharomyces cerevisiae and one strain of Saccharomyces carlsbergensis were arrested by the inhibitor of mitochondrial adenine nucleotide translocation, bongkrekic acid. This indicates that the mitochondrial adenine nucleotide translocation system is preserved and necessary for growth in a number of independent ρ⁻ mutants.

2. Growth of three “petite-negative” yeast species was arrested by a combined inhibition of respiration by antimycin A and of adenine nucleotide translocation by bongkrekic acid. Thus, the arrest of growth upon inhibition of adenine nucleotide translocation in non-respiring cells is not specific for ρ⁻ mutants and may be a general characteristic of eucaryotic cells.     

Transport in C4 mesophyll chloroplasts. Characterization of the pyruvate carrier

1. Evidence is presented for high rates of carrier-mediated uptake of pyruvate into the stroma of intact mesophyll chloroplasts of the C₄ plant Digitaria sanguinalis, but not the chloroplasts of the C₃ plant Spinacea oleracea. Uptake of pyruvate in the dark with the C₄ mesophyll chloroplasts was followed using two techniques: uptake of [¹⁴C]pyruvate as determined by silicon oil centrifugal filtration and uptake as indicated by absorbance changes at 535 nm (shrinkage/swelling) after addition of 0.1 M pyruvate salts.

2. Uptake of the pyruvate anion by an electrogenic carrier is suggested to be the major mode of transport. Chloroplast swelling was observed in potassium pyruvate plus valinomycin and uptake of [¹⁴C]pyruvate was inhibited by membrane-permeant anions. Valinomycin reduced uptake in the absence of external potassium and the inhibition could be reversed by addition of external potassium.

3. Uptake of pyruvic acid (or a pyruvate ⁻/OH⁻ antiport) is ruled unlikely since [¹⁴C]pyruvate uptake was relatively independent of the pH gradient across the envelope and addition of pyruvate to chloroplasts did not result in an alkalization of the medium. The low rate of swelling observed in ammonium pyruvate may be due to non-mediated permeation of pyruvic acid, which is possible only at high pyruvate concentrations.

4. The concentration of pyruvate in the stroma increased with external concentration over the range tested (up to 40 mM) but the concentration ratio (internal/external) was always less than one. The steady-state concentration of [¹⁴C]pyruvate in the stroma was dependent on the ionic strength of the medium, with saturation at roughly I = 0.04 M, while accumulation of the membrane-permeant cation tetraphenylmethylphosphonium decreased with increasing ionic strength. This suggests that ionic strength modifies a membrane potential (inside negative) across the envelope and that pyruvate uptake responds to the magnitude and direction of that potential (−80 mV at low ionic strength).

5. Chloride and inorganic phosphate were potent inhibitors of [¹⁴C]pyruvate uptake. Of the sulfhydryl reagents tested, N-ethylmaleimide was not inhibitory while mersalyl completely blocked [¹⁴C]pyruvate uptake and swelling in potassium pyruvate plus valinomycin. Pyruvate uptake, as measured by valinomycin induced swelling in potassium pyruvate, was highly temperature sensitive, with an energy of activation of 39 kcal/mol above 9 °C.

6. Phenylpyruvate, -ketoisovalerate, -ketoisocaproate, -cyano-4-hydroxycinnamic acid and -cyanocinnamic acid inhibited [¹⁴C]pyruvate but not [¹⁴C]-acetate uptake in the dark and also reduced pyruvate metabolism by the chloroplasts in the light.     

Properties and function of the respiratory chain of freshwater mussel spermatozoa in relation to motility

1. The spermatozoa of the freshwater mussel (Hyriopsis schlegelii) contain cytochromes aa₃, b and c, flavoproteins and nicotinamide nucleotides in molar ratios of 1.0:0.9:1.8:1.8:8.7. Cytochrome c₁ is not detectable even at liquid-N₂ temperature, but a c₁-like cytochrome with an -band at 550 mμ is found at liquid-N₂ temperature in a cell preparation from which cytochrome c is completely removed.

2. The near-ultraviolet difference spectrum of whole cells reveals an absorption peak at 315 mμ with a shoulder around 350 mμ.

3. Both the endogenous respiration and motility of spermatozoa are completely blocked by 0.2 mM CN⁻ and by 0.2 μM antimycin A. 2,4-Dinitrophenol and pentachlorophenol completely inhibit motility at the maximal stimulation of respiration. Rotenone strongly inhibits NADH oxidase of spermatozoa, although it has no effect on the respiration of whole cells.

4. It is concluded that the motility of mussel spermatozoa is tightly coupled to respiration, and the respiratory chain phosphorylating process is the only energy-supplying system for motility.     

The binding of aurovertin to mitochondria, and its effect on mitochondrial respiration

1. The fluorescence of aurovertin increases about 100-fold on binding to sub-mitochondrial particles.

2. The mitochondrial ATPase (F₁) binds one mole aurovertin/mole F₁ with a dissociation constant of 6·10⁻⁸ M.

3. The fluorescence of mitochondrion-bound aurovertin is maximal during State-3 respiration and is partially quenched on anaerobiosis, addition of respiratory inhibitor, oligomycin or uncoupler, or transition to State 4. This quenching is still present when the binding site is saturated with aurovertin, showing that the quantum yield of fluorescence is lowered.

4. Aurovertin is bound co-operatively to State-3 mitochondria.

5. The curve relating inhibition of State-3 respiration to aurovertin concentration is more sharply sigmoidal than the binding curve.

6. An analysis of the binding and inhibition data leads to the conclusion that aurovertin induces a conformation change in the binding site on F₁ in two ways: (i) directly by acting as an allosteric effector of an oligomeric system, (ii) indirectly by inhibiting State-3 respiration which changes the allosteric constant of the oligomeric system.

7. The concentration of the aurovertin-binding site in both rat-liver and rat-heart mitochondria is about the same as that of the antimycin-binding and oligomycin-binding sites.     

On the mechanism of oligomycin inhibition of Ca-induced mitochondrial respiration

The addition of oligomycin in the presence of Ca²⁺ increased the ADP pool in mitochondrial suspension. It is suggested that oligomycin inhibition of Ca²⁺-induced mitochondrial respiratory activation is the function of the increased endogenous ADP pool. Low ADP concentrations (5–20 μM) produce the same inhibitory effect as oligomycin. The increase of ADP levels in the presence of glucose plus hexokinase resulted in the inhibition of Ca²⁺-induced respiration, while the addition of phosphoenol pyruvate plus pyruvate kinase followed by a reduction in ADP levels, reversed the oligomycin inhibitory effect. One of the essential stages of ADP accumulation in mitochondrial suspensions in the presence of oligomycin and Ca²⁺ is proposed to be the formation of ADP from AMP and ATP, effected by adenylate kinase.     

On the nature of electron and energy transport in mitochondria. I. Multiple inhibition of mitochondrial respiration

1. A series of eight classical respiratory-chain inhibitors was studied. The slopes of State-3 respiratory rate versus dose plots are convex for antimycin, 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO), rotenone and sulfide, and concave for malonate, Amytal, cyanide and azide.

2. Plots of ADP: O ratio versus dose indicate uncoupling effects at higher concentrations of antimycin, HOQNO, cyanide and azide. On the other hand, sulfide and rotenone have no effect on the phosphorylating efficiency. Malonate increases the ADP: O ratio.

3. Two inhibitors can be combined in such a way that the total inhibition should be equal to the inhibition caused by the single inhibitors if each inhibitor affects respiration independently (additivity of inhibition). In practice, however, antagonism and synergism are also found.

4. Additivity of combined inhibition occurs where both inhibitors act on the same enzyme.

5. Antagonism is observed where the two inhibitors act on different enzymes of the same chain.

6. Synergism is found where the two inhibitors act on enzymes in different branches of a forked chain. This turns into normal additivity when the electron flow through both branches is made equal.

7. The results are compatible with the hypothesis that respiratory enzymes are arranged in chains. The possibility that the chains may be cross-linked or branched is discussed.     

Net movements of monovalent and bivalent cations, and their relation to energy metabolism, in slices of hepatoma 3924A and of a mammary tumour

1. During incubation at 1° in saline medium buffered either with phosphate or bicarbonate, slices of Morris hepatoma 3924A, and of a chemically induced tumour of rat mammary gland, lost K⁺ and gained Na⁺, Ca²⁺ and water.

2. Upon subsequent incubation at 38° in oxygenated medium, these changes were partially reversed. In the hepatoma, the reaccumulation of K⁺ was equally efficient in phosphate or bicarbonate medium, and in the presence and absence of glucose. Ca²⁺ was extruded in bicarbonate, but not in phosphate medium, and its extrusion was reduced in the presence of glucose.

3. When respiration was inhibited in the presence of glucose, K⁺ transport by the hepatoma continued to an extent which varied with the glycolytic activity of the slices, suggesting that the rate of ATP synthesis was a limiting factor under these conditions.

4. In the absence of glucose, the transport of Na⁺ and K⁺ was completely stopped by respiratory inhibition. However, more than 50% of the O₂ uptake had to be inhibited before any effect on transport was observed, suggesting that the rate of synthesis of ATP from endogenous respiration is in excess of that required to maintain transport.

5. Inhibition of transport by ouabain was accompanied by a 30% fall in the rate of endogenous respiration, and by a fall of 33% in the rate of glycolysis in the presence of cyanide plus glucose.

6. Comparison of the minimum rates of respiration and of glycolysis (in the presence of glucose plus cyanide) required to maintain the maximal extent of K⁺ transport in the hepatoma slices, suggests that ATP derived from oxidative phosphorylation or from anaerobic glycolysis is equally efficient as a source of energy for ion transport.     

Energy transduction in photosynthetic bacteria. XI. Further resolution of cytochromes of b type and the nature of the CO-sensitive oxidase present in the respiratory chain of Rhodopseudomonas capsulata

1. In membranes prepared from dark grown cells of Rhodopseudomonas capsulata, five cytochromes of b type (E′₀ at pH 7.0 +413±5, +270±5, +148±5, +56±5 and −32±5 mV) can be detected by redox titrations at different pH values. The midpoint potentials of only three of these cytochromes (b₁₄₈, b₅₆, and b₋₃₂) vary as a function of pH with a slope of 30 mV per pH unit.

2. In the presence of a Co/N₂ mixture, the apparent E′₀ of cytochrome b₂₇₀ shifts markedly towards higher potentials (+355 mV); a similar but less pronounced shift is apparent also for cytochrome b₁₅₀. The effect of CO on the midpoint potential of cytochrome b₂₇₀ is absent in the respiration deficient mutant M6 which possesses a specific lesion in the CO-sensitive segment of the branched respiratory chain present in the wild type strain.

3. Preparations of spheroplasts with lysozyme digestion lead to the release of a large amount of cytochrome c₂ and of virtually all cytochrome cc′. These preparations show a respiratory chain impaired in the electron pathway sensitive to low KCN concentration, in agreement with the proposed role of cytochrome c₂ in this branch; on the contrary, the activity of the CO-sensitive branch remains unaffected, indicating that neither cytochrome c₂ nor the CO-binding cytochrome cc′ are involved in this pathway.

4. Membranes prepared from spheroplasts still possess a CO-binding pigment characterized by maxima at 420.5, 543 and 574 nm and minima at 431, 560 nm in CO-difference spectra and with an band at 562.5 nm in reduced minus oxidized difference spectra. This membrane-bound cytochrome, which is coincident with cytochrome b₂₇₀, can be classified as a typical cytochrome “o” and considered the alternative CO-sensitive oxidase.     

Regulation of citrate metabolism and acetycholine synthesis by Ca in rat brain synaptosomes

The relationships between pyruvate and derived citrate metabolism and acetylcholine (ACh) synthesis in synaptosomes were examined. In the presence of 30 mM KCl, 0.1 mM Ca²⁺ caused 31 and 63% inhibition of pyruvate utilization and citrate accumulation, respectively. Verapamil and EGTA (0.5 mM) brought about no change in pyruvate consumption but increased rate of citrate accumulation, and overcame inhibitory effect of Ca²⁺. The rates of citrate accumulation in the presence of verapamil or EGTA were three to six times, respectively, higher than those in the presence of Ca²⁺. (−) Hydroxycitrate increased rate of citrate accumulation under all experimental conditions. The value of this activation appeared to be stable (0.20–0.28 nmol/min/mg of protein) and independent of changes in the basic rate of citrate accumulation. Ca²⁺ caused no significant changes in [¹⁴C]ACh synthesis, but it inhibited ¹⁴CO₂ production by synaptosomes. These activities were inhibited by verapamil by 33 and 60%, respectively. Ca²⁺ did not modify these effects of the drug. On the other hand, (−)hydroxycitrate resulted in 22 and 29% inhibition of [¹⁴C]ACh synthesis in Ca²⁺ free and Ca²⁺ supplemented medium, respectively. These data indicated that rates of acetyl-CoA synthesis in synaptoplasm, via ATP-citrate lyase and probably by another pathways are independent of Ca-evoked changes in pyruvate oxidation and citrate supply from intraterminal mitochondria. This property might play a significant role in maintenance of stable level of ACh in active cholinergic nerve endings.     

Insulin-like effects of fluoroacetate on lipolysis and lipogenesis in adipose tissue.

Hormone-stimulated lipolysis in adipose tissue was inhibited by fluoroacetate and there was a concomitant decrease in both the basal and hormone-stimulated cyclic AMP levels. Adenylate cyclase (EC 4.6.1.1) activity in membrane preparations was inhibited by fluoroacetate. There was no influence of fluoroacetate on the low Km cyclic AMP phosphodiesterase (EC 3.1.4.17) activity. The rate of glucose conversion to fatty acids was increased when adipose tissue was incubated in the presence of fluoroacetate. The outputs of pyruvate and lactate into the incubation medium were decreased at this time, suggesting decreased tissue pyruvate levels and a site of activation of lipogenesis distal to pyruvate formation. Pyruvate dehydrogenase (EC 1.2.4.1) activity was increased twofold in adipose tissue incubated in the presence of fluoroacetate. This was attributed to a fluoroacetate-induced inhibition of pyruvate dehydrogenase kinase, the enzyme responsible for inactivating the pyruvate dehydrogenase complex. Glucose transport was increased to a small but significant degree by fluoroacetate. In addition, both the tissue content of citrate and its release into the incubation medium were increased, suggesting that fluoroacetate resulted in an inhibition of aconitase (EC 4.2.1.3). The tissue ATP content was unchanged. Because the antilipolytic and lipogenic effects of fluoroacetate parallel those of insulin, they may share a common mechanism.     

Mitochondrial functions under hypoxic conditions: The steady states of cytochrome c reduction and of energy metabolism

1. The effect of low oxygen concentration on the oxidation-reduction states of cytochrome c and of pyridine nucleotide, on Ca²⁺ uptake, on the energy-linked reduction of pyridine nucleotide by succinate, and on the rate of oxygen consumption have been examined under various metabolic conditions, using pigeon heart mitochondria.

2. The oxygen concentration required to provide half-maximal reduction of cytochrome c (p50_c) ranges from 0.27 to 0.03 μM (0.2-0.02 Torr) depending upon the metabolic activity. There is a linear increase of the p50_c value with increasing respiratory rate.

3. The fraction of the normoxic respiration that is observed at p50_c is 70–90% under State 4 conditions, but is 30% under State 3 conditions.

4. The oxygen requirement for half-maximal reduction of pyridine nucleotide (p50_PN) varies less than p50_c, being 0.08 μM in State 3 and 0.06 μM in the uncoupled state.

5. The ability of the mitochondria to exhibit an energy-linked reduction of pyridine nucleotide by succinate disappears at an oxygen concentration of 0.09 μM (0.06 Torr). Below this oxygen concentration, endogenous Ca²⁺ begins to be released from the mitochondria. Thus, the critical oxygen concentration for bioenergetic function of mitochondria corresponds approximately to 50% reduction of pyridine nucleotide (p50_PN).     

The mechanism of inhibition by oligomycin of oxidative phosphorylation in mitochondria

1. The concentration of specific oligomycin-binding sites in rat-liver mitochondria is 0.12 nmole/mg protein, whereas at least 10-times more oligomycin can be bound non-specifically.

2. The activity of oligomycin-inhibited processes in intact mitochondria and submitochondrial particles cannot be restored by treatment with egg lecithin or mitochondrial phospholipids.

3. Analysis of the kinetics of inhibition of State-3 respiration by oligomycin reveals that (i) after a certain lag period the inhibition by oligomycin is pseudo-first order with respect to the respiratory-control ratio, defined as the ratio of the respiratory rate at time t to that of the final inhibited site, (ii) the value of the pseudo-first-order rate constant (k₀) is dependent on the oligomycin: protein ratio, phospholipid: protein ratio, pH and temperature, (iii) the effects of various substrates and inhibitors of electron transfer on the kinetics of oligomycin inhibition can be explained by their effects on respiratory control.

4. A detailed model is proposed for the interaction of oligomycin with mitochondria. It is proposed that two conformations of the oligomycin-sensitive site are present, and that oligomycin specifically binds to the conformation that is involved in the induction of respiratory control.     

The effect of mersalyl on the oxidation of succinate by housefly mitochondria

1. Succinate is oxidized very rapidly by housefly flight-muscle mitochondria in the presence of rotenone.

2. Phosphate (or arsenate) is a strong inhibitor of this succinate oxidation.

3. The inhibitory effect of phosphate (or arsenate) can be overcome by mersalyl and other sulphydryl reagents.

4. Under conditions of rapid succinate oxidation the intramitochondrial phosphate concentration is low.

5. It is suggested that phosphate competes with succinate for entry into the mitochondria.     

Porphyrin-sensitized photodynamic damage of isolated rat liver mitochondria

The respiration rates and the respiratory control ratios of isolated rat liver mitochondria have been measured following exposure to 0–160 kJ/m² of near-ultraviolet radiation (blacklight) in the presence of low concentrations of porphyrins (0.1–0.2 μmol/l).

Depending on the light dose, the concentration and the type of porphyrin, the following sequence of reactions occurred: uncoupling and inhibition of oxidative phosphorylation, energy dissipation, inhibition of respiration and swelling and disruption of the mitochondria.

The detrimental effects could not be elicited in the absence of oxygen, neither could they be elicited by porphyrins or light alone.

At equimolar concentrations, the effectiveness of the porphyrins as photosensitizers were: deuteroporphyrin > protoporphyrin coproporphyrin > murophorphyrin.

The results may be of importance to explain the skin lesions seen when porphyrins of different hydrophobicity accumulate in the skin.     

Respiratory control in submitochondrial particles obtained by sonication

1. Uncoupling agents markedly stimulate oxidation of NADH and succinate by particles obtained from sonication of heavy beef-heart mitochondria. Such respiratory stimulation is demonstrable in the complete absence of factors or agents affecting the phosphorylation sequence itself.

2. The respiratory control thus revealed is most prominent at the NADH-flavin coupling site but is also present at the cytochrome b region coupling site.

3. Uncoupler concentrations inducing maximal respiratory rates exceed those abolishing the phosphorylative capacity by one order of magnitude or more.

4. The addition of glucose, hexokinase and ADP prior to that of uncoupler reduces the uncoupler-induced respiratory stimulation.

5. A respiratory stimulation initiated by Ca²⁺ is additive to the uncoupler-nduced effect both in the NADH and succinate oxidase systems.