Presence Of Rotenone Research Articles

The decrease of mitochondrial substrate uptake caused by trialkyltin and trialkyl-lead compounds in chloride media and its relevance to inhibition of oxidative phosphorylation.

1. In a 100 mM-KCl medium (pH 6.8) containing ATP, triethyltin (1 muM) causes a decrease in the uptake of pyruvate, malate, citrate or beta-hydroxybutyrate by rat liver mitochondria, but no decrease is observed in a 100 mM-KNO3 medium. This response is not modified by the presence of rotenone in the incubation medium. 2. In the KCl medium at least 1 muM-triethyltin is required to cause maximum inhibition of pyruvate uptake. 3. Trimethyltin, tributyltin and the trialkyl-lead analogues at 1 muM, to varying degrees, also cause a decrease in pyruvate uptake by mitochondria only in the KCl medium. 4. Triethyltin stimulates resting respiration of mitochondria with all the substrates tested in the KCl medium but not in the KNO3 medium, yet this stimulation of O2 uptake occurs under conditions when substrate uptake is decreased. 5. In contrast, both O2 uptake during state 3 respiration and ATP synthesis when linked to the oxidation of pyruvate, malate or citrate are strongly inhibited by 1 muM-triethyltin in a KCl medium, but O2 uptake and ATP synthesis during the oxidation of beta-hydroxybutyrate are only slightly affected. In a KNO3 medium O2 uptake and ATP synthesis linked to the oxidation of all substrates are only slightly affected. 6. The relevance of the decrease in substrate uptake by mitochondria caused by triethyltin in a KCl medium to the greater sensitivity of various mitochondrial functions observed in vitro is discussed. It is concluded that decrease of matrix substrate content is probably not the major cause of the greater sensitivity of oxidative phosphorylation to triethyltin in a KCl medium observed previously.

The penetration of the membrane of brain mitochondria by anions.

Abstract—The permeability of the membrane of rat brain non‐synaptosomal mitochondria, towards inorganic and substrate anions, was assessed by measuring the rate of swelling that occurred when mitochondria were suspended in an iso‐osmotic solution of a permeant anion, in the presence of a permeant cation such as NH+4 or K+ in the presence or absence of valinomycin. In NH+4‐phosphate swelling was higher than it was in KCI or K+‐phosphate, which showed the prevalence of the mechanism of phosphate transport previously demonstrated in liver mitochondria. The entry of succinate and L‐malate seemed to require the presence in the inner mitochondrial membrane of specific carriers. as previously postulated for liver mitochondria, but the rate of swelling of brain mitochondria was lower than that of liver organelles. In K+‐succinate, in the presence of antimycin, added ATP induced swelling and this was attributable to the simultaneous permeation both of the anion and the cation. Fumarate did not penetrate into brain mitochondria. Practically no swelling was recorded in NH+4 or K+‐citrate, which indicated that this anion penetrated poorly into the isolated brain mitochondria even in the presence of malate. Swelling occurred in NH+4‐L‐glutamate in the presence of rotenone, and the entry of this anion seemed to follow a gradient of concentration although the presence of a specific translocator in the inner mitochondrial membrane might be concerned. The entry of glutamate was independent of that of phosphate and N‐ethylmaleimide appeared to be a specific inhibitor of this entry. Swelling in K+‐L‐glutamate, in the presence of rotenone, was enhanced by the addition of valinomycin or ATP but in the latter case when osmotic equilibrium was reached swelling was not reversed by oligomycin. In conclusion, the lesser extent of swelling of isolated brain mitochondria compared with liver mitochondria could be attributed to the heterogeneity of the populations of these organelles, each population possessing its own characteristics of membrane permeability. Observations of electron micrographs of brain mitochondria incubated in iso‐osmotic substrate anions confirmed the heterogeneous rate of swelling of these particles.

Calcium Accumulation by Chondrocyte Mitochondria

Chick epiphyseal plate mitochondria observed in vitro suggest that energy dependent Ca++ uptake was maximal in the presence of ATP and a respiratory substrate. However, nucleotides other than ATP had no effect on this type of cation uptake. The observation that Ca++ accumulation was sensitive to the presence of 2,4-DNP and a number of respiratory inhibitors suggested that the mechanism of cation accumulation was similar to that described in tissues that do not undergo biological mineralization. Non-energy supported Ca++ acumulation was studied in the presence of rotenone and antimycin A. Under these conditions, the amount of Ca++ bound by skeletal tissue mitochondria was greater than bound by mitochondria obtained from noncalcifying tissues. Following isopycnic centrifugation, the Ca++ loaded mitochondria banded at different sucrose densties but the Ca++ affinity of mitochondria at each density band was similar. Hence, no particular mitochondrial species seems to be responsible for cation transport.

The Influence of Respiration and ATP Hydrolysis on the Proton‐Electrochemical Gradient across the Inner Membrane of Rat‐Liver Mitochondria as Determined by Ion Distribution

A technique is described, based on the distribution of rubidium, acetate and methylammonium ions, for the simultaneous estimation of membrane potential and pH gradient across the inner membrane of mitochondria. The technique requires less than 0.5 mg mitochondrial protein and is independent of many factors which interfere with electrode determinations of protonmotive force (Δp). With a limiting matrix volume of 0.4 μl/mg mitochondrial protein, the indicated value of Δp for rat liver mitochondria is 228 mV in state 4, 170 mV in state 3, and –0.6 mV in the presence of rotenone and uncoupler. The relative contributions of the pH gradient and membrane potential are dependent on the availability of electrophoretically and electroneutrally translocatable species in the incubation medium. In a sucrose‐based medium containing 0.5 mM KCl, rotenone and uncoupler, the technique indicated a membrane potential of + 85 mV and a pH gradient of + 1.46 (acidic in the matrix compartment). In state 4, under no conditions examined did the pH gradient contribute more than 50% of the total protonmotive force. The hydrolysis of ATP generates an optimal Δp of 220 mV. The proton conductance of the inner membrane is potential dependent, increasing when Δp is greater than 200 mV. The extra‐mitochondrial phosphate potential sustainable by respiration was found to change in parallel to Δp, but to exceed the latter parameter when based upon a stoichiometry of two protons translocated per ATP synthesised.

Uptake of calcium ions by synaptosomes from rat brain

Rat brain synaptosomes incubated in modified Krebs-Ringer media accumulated 45Ca in amounts dependent upon the medium calcium concentration. At 37 °C, 45Ca uptake was increased by 19% upon the application of electrical pulses. Uptake was effectively inhibited by ruthenium red, uncouplers of oxidative phosphorylation, antimycin A and rotenone, and fluorocitrate while oligomycin and ouabain increased 45Ca uptake. Transfer to a medium low in Na + led to marked increase in 45Ca uptake and this effect was also found in the presence of rotenone and arsenate. The results indicate the requirements of metabolic energy for a component of calcium uptake by synaptosomes. Effects of low Na + incubation and electrical stimulation may be due to events at the outer synaptosomal membrane though the possibility of direct effects at the mitochondrion have not been excluded.

The effects of acetylcolletotrichin on the mitochondrial respiratory chain.

1. Acetylcolletotrichin is a phytotoxic compound that has been isolated from the culture medium of the fungus Colletotrichum capsici (Grove et al., 1966). 2. With isolated liver and kidney mitochondria acetylcolletotrichin markedly inhibited the oxidation of succinate and those substrates with NAD-linked dehydrogenases, but did not inhibit the oxidation of ascorbate in the presence of tetramethyl-p-phenylenediamine. In this respect its action was similar to that of antimycin A. 3. Acetylcolletotrichin differed from antimycin in that, even at high concentrations which produced a maximal inhibitory effect, its action was partially reversed by uncoupling agents. Also acetylcolletotrichin had no detectable effect on the oxidative activity of blowfly flight-muscle mitochondria and was not very effective with heart mitochondria. 4. Acetylcolletotrichin inhibited the oxidative activity of liver mitochondria more markedly when respiration was stimulated by ADP together with phosphate and was less effective when respiration was stimulated by uncoupling agents. 5. There was an unusual interaction between the succinate oxidation system and the oxidation of glutamate together with malate. Thus, glutamate together with malate, even in the presence of rotenone, markedly decreased the effectiveness of acetylcolletotrichin in inhibiting succinate oxidation. 6. These effects were paralleled in the observed redox changes of cytochrome c. 7. The unusual behaviour of the cytochromes b in the presence of acetylcolletotrichin is described, and it is suggested tentatively that this inhibitor acts between cytochromes b with absorption maxima at 30 degrees C of approximately 560 and 565nm.

Oxaloacetate inhibition of succinate oxidation in tightly coupled liver mitochondria with ferricyanide as an electron acceptor

When ferricyanide is used as an artificial electron acceptor, succinate oxidation by tightly coupled liver mitochondria becomes inhibited after 1–3 min. No inhibition occurs in the presence of rotenone or glutamate establishing that oxaloacetate causes the inhibtion. Oxygen consumption by mitochondria oxidizing succinate does not become inhibited in the absence of rotenone suggesting that oxaloacetate accumulates to a greater extent when ferricyanide is added than when oxygen is the terminal acceptor. Higher levels of oxaloacetate in the ferricyanide reaction are apparently due to an increased rate of synthesis rather than a decreased rate of removal. Thus it appears that when succinate is the substrate and oxygen the terminal acceptor a control mechanism exists which blocks oxidation of malate. When ferricyanide is added as an artificial electron acceptor this control is lost and oxaloacetate accumulates to inhibit succinate oxidation.

Calcium transport in intact ehrlich ascites tumor cells

1.1. Ca2+ transport by both intact ascites tumor cells and ascites cell mitochondria was studied spectrophotometrically using murexide, a metallochronic indicator of Ca2+ concentrations.2.2. Ascites tumor cells can accumulate in vitro up to 30 μmoles of Ca2+ per g dry wt within 3–4 min. The accumulation occurs when endogenous or added mitochondrial substrates provide for a coupled respiration. By contrast, glycolysis is unable to support Ca2+ uptake.3.3. In the presence of succinate and rotenone, ascites tumor cells are able to accumulate and retain significant amounts of Ca2+. In the presence of endogenous or added NADH-linked substrates, the uptake of Ca2+ was less and in a few minutes all the Ca2+ accumulated by the cells is released into the medium. This release is specific for Ca2+ and does not occur with Mr2+ and Sr2+, which are accumulated and retained by the cells.4.4. Respiration of ascites cells was stimulated by the addition of Ca2+. In the presence of rotenone and succinate, the rate of respiration returns to basal levels after the Ca2+ has been transported into the cell. However, with other substrates, Ca2+ causes a stimulation of the respiration which continues even after the accumulation and subsequent release of Ca2+.5.5. Ca2+ uptake by ascites cells was only slightly affected by the variation of monovalent cations, pH or osmolarity of the medium. It was completely inhibited by the addition of 10 mM Mg2+ or 15 μM ruthenium red.6.6. The results obtained indicate that ascites tumor cells possess a plasma membrane permeable to Ca2+ and that the Ca2+ uptake by mitochondria inside the cell accounts for the entire cellular Ca2+ accumulation. The use of ascites cells as a model for studying mitochondrial Ca2+ uptake in vivo and the metabolic aspects involved in Ca2+ transport are discussed.

Calcium and pyridine nucleotide interaction in mitochondrial membranes

The interaction of NADH with divalent cations was studied in model systems and in isolated rat liver mitochondria. The addition of Ca 2+ to solutions of NADH in methanol, but not in water, produces a large increase in fluorescence intensity and a shift in the emission spectrum to a shorter wavelength. The ratio of fluorescence intensity in the presence of Ca 2+ with respect to that in the absence of Ca 2+ is 1 in aqueous and 2.4 in methanol solutions. Other di- or trivalent cations produce a similar enhancement of NADH fluorescence in nonpolar solutions, except for Mn 2+ which quenches the fluorescence of NADH. The energy-dependent accumulation of Ca 2+ in mitochondria increases the NADH fluorescence in the presence of rotenone. This effect was absent when Mn 2+ was previously accumulated by mitochondria. With the NADH-linked substrates, but not with succinate and rotenone, the accumulation of large amounts of Ca 2+ in mitochondria causes a depression of respiration and a release of the accumulated Ca 2+. The inhibition of the respiration is accompanied by an increased permeability and a depletion of the endogenous pyridine nucleotides. Both effects were not directly related to the concomitant mitochondrial swelling. On the basis of this data, the formation of a complex between calcium and NADH in the nonpolar region of the mitochondrial membrane is proposed. The physiological implications of this complex are discussed.

Interrelationship and control of glucose metabolism and lipogenesis in isolated fat-cells. Control of pentose phosphate-cycle activity by cellular requirement for reduced nicotinamide adenine dinucleotide phosphate.

By using inhibitors and stimulators of different metabolic pathways the interdependence of the pentose phosphate cycle and lipogenesis in isolated fat-cells was studied. Rotenone, which is known to inhibit electron transport in the respiratory chain, blocked glucose breakdown at the site of pyruvate dehydrogenase. Consequently, because of the lack of acetyl-CoA, fatty acid synthesis was almost abolished. A concomitant decrease in pentose phosphate-cycle activity was observed. Phenazine methosulphate stimulated pentose phosphate-cycle activity about five- to ten-fold without a considerable effect on fatty acid synthesis. The influence of rotenone on both the pentose phosphate cycle and lipogenesis could be overcome by addition of phenazine methosulphate, indicating that rotenone has no direct effect on these pathways. The decreased rate of the pentose phosphate cycle in the presence of rotenone therefore has to be considered as a consequence of decreased fatty acid synthesis. The rate of glucose catabolism via the pentose phosphate cycle in adipocytes appears to be determined by the requirement of NADPH for lipogenesis. Treatment of cells with 6-aminonicotinamide caused an accumulation of 6-phosphogluconate, indicating an inhibition of 6-phosphogluconate dehydrogenase. The rate of glucose metabolism via the pentose phosphate cycle as well as the rate of fatty acid synthesis, however, was not affected by 6-aminonicotinamide treatment and could still be stimulated by addition of insulin. Since even in cells from starved animals, in which the pentose phosphate-cycle activity is extremely low, no accumulation of 6-phosphogluconate was observed, it is concluded that the control of this pathway is achieved by the rate of regeneration of NADP at the site of glucose 6-phosphate dehydrogenase.

Studies on the energy state of isolated brown adipose tissue mitochondria the cytochrome b complex as a probe of the energy state of the mitochondrial inner membrane

Brown adipose tissue mitochondria from cold-stressed guinea pigs isolated in sucrose-HEPES-EDTA medium (HEPES = N-2-hydroxyethylpiperazine- N′-2-ethanesulphonic acid) possess several criteria of loose coupling (J. I. Pedersen and H. J. Grav, Eur. J. Biochem., 25 (1972) 75), and the present studies on the steady-state oxidation-reduction level of the cytochrome b complex under various experimental conditions have confirmed this conclusion. Generally, the kinetics as well as the steady-state redox level of b-type cytochromes in brown adipose tissue mitochondria differ markedly from those of coupled liver mitochondria of the same animals. The main features of these mitochondria are: 1. 1. In the presence of rotenone, cyanide, ascorbate, and N, N, N′, N′-tetramethyl- p-phenylenediamine (TMPD) the addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) induces no oxidation of b-type cytochromes in brown adipose tissue mitochondria whereas a 30% oxidation was observed with liver mitochondria. This fact indicates that freshly isolated brown adipose tissue mitochondria are completely deenergized. 2. 2. The percentage degree of reduction of b 561 by succinate increases almost linearly from pH 6.0 (approx. 10%) to pH 8.0 (approx. 32%) in brown adipose tissue mitochondria, whereas the reduction of b 561 in liver mitochondria (approx. 16%) is almost independent of pH in the same region. 3. 3. With brown adipose tissue mitochondria, but not with liver mitochondria, the subsequent reduction of b 565 by ATP was dependent on added P i. 4. 4. The ATP-induced reduction of b 565 in liver mitochondria was completely inhibited by oligomycin whereas 87% inhibition was observed with brown adipose tissue mitochondria. The uncoupler FCCP, however, completely inhibited the ATP response. The oligomycin-insensitive reduction could also be induced by other highenergy nucleotides such as GDP and GTP. 5. 5. The ATP-induced reduction of b 565 in brown adipose tissue mitochondria revealed a pH dependence very different from that of liver mitochondria; the pH optium was around 7.4 in liver mitochondria whereas that of brown adipose tissue mitochondria was approx. 1 pH unit lower. 6. 6. The concentration of ATP needed for half-maximal reduction of b 565 was 30 times higher with brown adipose tissue mitochondria than with liver mitochondria under identical experimental conditions. 7. 7. The significance of the pH and the intramitochondrial concentrations of adenine nucleotides and phosphate in the regulation of the coupling of respiration to phosphorylation in brown adipose tissue mitochondria is discussed.

Fluorescence of nicotinamide adenine dinucleotides during the active transport of Ca 2+ ions in liver mitochondria.

Accumulation of Ca2+ ions by rat liver mitochondria increases the fluorescence of the reduced pyridine nucleotides in the presence of rotenone. The fluorescence increase is sensitive to the uncouplers of the oxidative phosphorylation and to the inhibitors of the electron transfer, providing the release of the accumulated Ca2+ from mitochondria. The substantial change of the fluorescence occurs in the presence of acetate, when the accumulated Ca2+ is present in the intramitochondrial space as soluble salt. Addition of Ca2+ to water solution of NADH and NADPH is followed by the slight increase of the fluorescence, while the same nucleotides dissolved in the more hydrophobic medium (methanol) considerably increase the fluorescence level by addition of Ca2+. The increase of the fluorescence of NAD(P)H in the mitochondria due to the accumulation of Ca2+ is considered not to be caused by the alkalinization of the intramitochondrial space but by the formation of the nucleotide-metal complex, localized at least partly at a hydrophobic phase.

The oxidation of malate by isolated plant mitochondria.

The functional relationship between NAD+‐linked oxidations of L‐malate and the operation of the exogenous and endogenous NADH dehydrogenases of isolated plant mitochondria have been investigated. Mitochondria isolated from tubers of the Jerusalem artichoke (Helianthus tuberosus) oxidised L‐malate via an electron transport pathway which was inhibited by rotenone, piericidin A and added oxaloacetate. Phosphorylation linked to this pathway yielded an APD/O ratio of approx. 3. In the presence of rotenone, piericidin A or added oxaloacetate, malate was oxidised by a pathway which was dependent on the presence of added NAD+ and low levels of Ca2+. Phosphorylation linked to this oxidation had an ADP/O ratio of approx. 2. Oxidation of malate via the rotenone‐sensitive pathway was inhibited by 2‐n‐butylmalonate whereas oxidation via the rotenone‐insensitive pathway was unaffected by this compound. By following the reduction of ferricyanide by malate in intact mitochondria, it was possible to identify two separate malate: NAD oxidoreductases: one located outside the inner membrane permeability barrier, the other inside this barrier. Oxaloacetate was the primary product when malate was oxidised by the enzyme in the inner compartment which was therefore identified as L‐malate: NAD oxidoreductase whereas pyruvate was the primary product of malate oxidation by the enzyme in the outer compartment, which was identified as L‐malate : NAD oxidoreductase (decarboxylating) or malic enzyme. The malic enzyme was extracted and partially purified from isolated mitochondria. The isolated enzyme showed a requirement for Mn2+ or Mg2+, required NAD+ as coenzyme, although activity to a lesser extent was obtained with NADP+. and was inhibited by NADH. It was concluded that in intact plant mitochondria there were two pathways by which malate can be oxidised: in the inner compartment via malate dehydrogenase in association with the endogenous NADH dehydrogenase system and in the intermembrane compartment via NAD+‐linked malic enzyme associated with the exogenous NADH dehydrogenase system. The implications these findings are discussed. L‐Malate is poorly oxidised by malate dehydrogenase in animal mitochondria unless the oxaloacetate formed as a product is removed, by either transamination with glutamate or by condensation with acetyl‐CoA [1–3]. In contrast isolated plant mitochondria will rapidly oxidise malate in the absence of added oxaloacetate‐removing agents [4–7].The formation of some pyruvate during the oxidation of malate has been observed in avocado fruit mitochondria [9] and apple peel mitochondria [10] and increased rates of malate oxidation have been reported in the presence of CoA and thiamine pyrophosphate [8–9], suggesting that pyruvate gave rise to acetyl‐CoA which could remove the oxaloacetate formed. Recently it was shown that in cauliflower bud mitochondria, pyruvate was a direct product of malate oxidation by a NAD+‐requiring malic enzyme, which showed a dependence on added NAD+ and was linked to the electron transport chain [11].

The effect of ATP on the EPR spectrum of phosphorylating sub-mitochondrial particles

1. The addition of either ATP or antimycin to substrate-reduced phosphorylating sub-mitochondrial particles causes a decline in the intensity of the EPR signals derived from free radicals and the iron-sulphur paramagnetic signals. 2. It is suggested that antimycin induces a conformation change, resulting in the redistribution of electrons within the paramagnetic centre. 3. It is suggested that ATP, on the other hand, causes the oxidation of iron-sulphur proteins. Two species are involved, one with g z = 2.02, g y = 1.94 and g x = 1.92, and one with g ⊥ = 1.94 and a g  of low intensity. With NADH as substrate, the main effect of ATP is to cause oxidation of the latter species; with succinate (in the presence of rotenone) the main effect is on the former species. 4. In the presence of antimycin, with NADH as substrate, ATP causes the oxidation of both species. With succinate as substrate (in the presence of rotenone) ATP has a much smaller effect on the g = 1.94 line in the presence of antimycin than in its absence. 5. In the presence of antimycin (but not in its absence) the addition of ATP causes the appearance of the g = 6 signal, characteristic of a 3 in partially reduced cytochrome aa 3, as well as the g ⊥ line of Cu(II). It is concluded that, when reversal of the chain is inhibited by antimycin, ATP induces a displacement of electrons within the cytochrome c oxidase molecule that results in a 3 being more oxidized than a. 6. It is pointed out that, despite the fact that 15 or 16 electron carriers have been identified in the respiratory chain, there still remain unidentified electron sinks where electrons disappear on adding ATP and reappear on adding uncoupler.

The redox states of respiratory-chain components in ratliver mitochondria. III. ‘Cross-over points’ in site III

1. When ascorbate is used as a substrate for rat-liver mitochondria in the presence of rotenone, antimycin and N, N, N′, N′-tetramethyl- p-phenylenediamine or phenazine methosulphate, a cross-over point is seen between cytochrome a 3 and oxygen. 2. This is shifted to between cytochromes c and a or below cytochrome c on adding varying amounts of terminal respiratory inhibitors. 3. With low substrate concentrations, positive cross-over points were observed between cytochromes c and a and between cytochromes a 3 and oxygen, and a negative cross-over between cytochromes a and a 3. 4. The cross-over data are consistent with a mechanism for Site-III phosphorylation that involves a high-potential form of one carrier (maybe copper) and a low-potential form of a 3, analogous to the mechanism of Site-II phosphorylation previously proposed.

Effect of phenoxybenzamine on the respiration of isolated rat liver mitochondria

The effect of phenoxybenzamine added in vitro to isolated rat liver mitochondria was investigated. Respiration was measured polarographically with either glutamate and malate or succinate as substrate in the presence of rotenone. Phenoxybenzamine inhibited the ADP-deficient (State 4), the ADP-activated (State 3), and the uncoupled (2,4-dinitrophenol, 40 μ m) respiration with all substrates used. The inhibitory action of phenoxybenzamine was dependent on the amount of mitochondrial protein present in the reaction medium. The higher the mitochondrial concentration, the higher the concentration of phenoxybenzamine required for inhibition. Serum albumin up to 1.4 mg/ml did not alter the effect of phenoxybenzamine. The inhibition of respiration was not competitive with succinate.

EPR studies on the iron-sulfur centers of dpnh dehydrogenase during the redox cycle of the enzyme

Summary Preparations of the inner mitochondrial membrane, upon treatment with DPNH in the aerobic state, undergo a cycle of absorbance changes, which may be monitored at 470–500 mμ. There is rapid initial bleaching, followed by return of the color on exhaustion of DPNH. In the presence of rotenone or piericidin the cycle is prolonged, the return of color (reoxidation) is inhibited and the extent of reoxidation less than in uninhibited particles. The EPR signals of four different iron-sulfur centers associated with DPNH dehydrogenase have thus far been resolved at temperatures between 4–20°K. Of these the iron-sulfur center of lowest potential, center 1, is reoxidized by the respiratory chain when DPNH is exhausted, but the high potential center 2 remains reduced. ATP reoxidizes iron-sulfur center 2 and restores full color to the chromophore. It is suggested that the energy for the reoxidation of the chromophore and iron-sulfur center 2 is supplied by coupling site I. On the basis of these and of other observations it is tentatively proposed that coupling site I is directly associated with DPNH dehydrogenase and is located on the 0 2 side of iron-sulfur center 1 and the substrate side of both center 2 and the specific binding sites of rotenone and piericidin.

Effects of o-phenanthroline and rotenone on the energy-linked swelling of rat liver mitochondria

Swelling of rat liver mitochondria can be mediated by energy conservation at any of the three sites. The presence of rotenone did not inhibit the energy-linked swelling when linked to the first site. On the other hand, o-phenanthroline completely eliminated this swelling. That the complete lack of swelling was due to inhibition of energy conservation at site I was established using swelling mediated specifically by energy conservation at site III. These results indicate that the energy conservation site I is located between the non-heme iron associated with NADH dehydrogenase and the rotenone sensitive site in mitochondria.

Phosphate transport in rat-liver mitochondria

1. The kinetics of the efflux of P i and malate as well as the relationship between P i transport and intra- and extramitochondrial pH changes were studied in rat-liver mitochondria in the presence of rotenone and oligomycin at different pH's. 2. At high pH a fast efflux of P i from the mitochondria occurs in the first few seconds, followed by a slow re-entry of P i into the mitochondria. Under the same conditions the exit of malate shows a time lag of 2–4 sec. The exit of malate coincides with the re-entry of P i. 3. In the presence of butylmalonate the exit of endogenous P i is coupled with a concomitant alkalinization of the mitochondrial matrix space, as calculated from the distribution of 5,5-[ 14C]dimethyloxazolidine-2,4-dione. 4. The stoicheiometry of the P i-hydroxyl exchange was found to be 1:1. 5. The kinetics of P i transport are consistent with previous observations that there is a direct exchange between OH − and P i, but not between OH − and malate. The equilibrium distribution of H 2PO 4 − and OH − deviates from the Donnan distribution. This may be explained by assuming a pH-dependent binding of P i in the mitochondria.

High-energy forms of cytochrome b. I. The effect of ATP and antimycin on cytochrome b in phosphorylating sub-mitochondrial particles

1. As in non-phosphorylating particles, antimycin causes a shift of the wavelength of maximum absorption of ferrocytochrome b in phosphorylating particles to the red by 2 nm. The antimycin-effect curve is sigmoidal. 2. ATP also causes a red shift, less than that brought about by antimycin and additive to the effect of the latter. 3. In addition to a red shift ATP brings about an oxidation of cytochrome b. 4. The ATP-induced red shift also occurs in the presence of rotenone when NADH is electron donor and in the presence of malonate or 4,4,4-trifluoro-1-(2-thionyl)-1,3-butadione when succinate is the donor. 5. The ATP-induced oxidation of cytochrome b occurs in the presence of both rotenone and antimycin. The oxidant is probably ubiquinone. 6. It is proposed that mitochondria contain two species of cytochrome b in equal amounts, one ( b i) affected by antimycin and the other ( b) not affected. Antimycin reacts preferentially with a form of b i present in high concentrations in energized particles. 7. It is proposed that the ATP-induced red shift is due to the formation of b 2+ ∼ X, a low-potential cytochrome b ( b 2+ ∼ X ag b 3+ + X + e). 8. It is proposed that b i 3+ ∼ X is a high-potential cytochrome b ( b i 2+ + X ag b i 3+ ∼ X + e). 9. A mechanism of Site-II phosphorylation is proposed in which ATP synthesis is linked with intramolecular electron transfer within the dimer b 2+ ∼ X · b i 3+ ∼ X.