Inhibition of energy-transducing functions of chloroplasts by spegazzinine.

The effects of spegazzinine, a dihydroindole alkaloid, on various energy-transducing functions of chloroplasts were studied. The following observations were made, (i) Spegazzinine inhibited both cyclic and noncyclic photophosphorylation in isolated spinach chloroplast. The I₅₀ value was about 80 μm. Over a concentration range which gave marked inhibition of ÀTP synthesis, there was no effect on basal or uncoupled electron flow or light-induced proton accumulation by isolated thylakoids, while the fraction of electron transport stimulated by coupled phosphorylation was reduced to the basal level by spegazzinine. (ii) The regulatory effect of low concentrations of ATP on proton movements and electron transport was diminished by the alkaloid, (iii) Spegazzinine also inhibited with similar efficiency the ATPase activities of membrane-bound coupling factor 1 (CF₁) and of purified CF₁. One mole of spegazzinine per mole of CF₁ seemed to be required to inhibit the ATPase activity, (iv) The allosteric effect of ADP on ATPase activity was not affected by spegazzinine. (v) On the basis of these results it is concluded that spegazzinine acts as an energy transfer inhibitor of hotophosphorylation and that its site of action may be at or near the catalytic site of ATPase.     

Cubebin and derivatives as inhibitors of mitochondrial complex I. Proposed interaction with subunit B8

The effects on mitochondrial respiration and complex I NADH oxidase activity of cubebin and derivatives were evaluated. The compounds inhibited the state 3 glutamate/malate-supported respiration of hamster liver mitochondria with IC₅₀ values ranging from 12.16 to 83.96 μM. NADH oxidase reaction was evaluated in submitochondrial particles. The compounds also inhibited this activity, showing the same order of potency observed for effects on state 3 respiration, as well as a tendency towards a non-competitive type of inhibition (K_I values ranging from 0.62 to 16.1 μM). A potential binding mode of these compounds with complex I subunit B8, assessed by docking calculations, is proposed.     

Effects of quercetin and F1 inhibitor on mitochondrial ATPase and energy-linked reactions in submitochondrial particles

Quercetin (3,3′,4′,5,7-pentahydroxyflavone) shares certain properties with the mitochondrial ATPase inhibitor protein. At low concentrations it inhibits both soluble and particulate mitochondrial ATPase and has no effect on oxidative phosphorylation in submitochondrial particles. Unlike the mitochondrial inhibitor protein quercetin inhibits the ATP-dependent reduction of NAD⁺ by succinate in fully reconstituted submitochondrial particles. A comparison of various flavones indicates that the hydroxyl groups at the 3′ and perhaps 3 position are important for the inhibition of ATPase activity.     

Action of the adenosine triphosphate analog, adenylyl imidodiphosphate in mitochondria.

Adenylyl imidodiphosphate (AMP-PNP), and analog of adenosine triphosphate (ATP), is a potent competitive inhibitor of mitochondrial ATPase activity. It inhibits both the soluble oligomycin-insensitive ATPase (K_i = 9.2 × 10^?7 M) and the bound oligomycin-sensitive APTase (K_i = 1.3 × 10^?6 M). ATPase activity of inside-out submitochondrial preparations are more sensitive to AMP-PNP in the presence of an uncoupler (K_i = 2.0 × 10^?7 M). Mitochondrial ATP-dependent reactions (reversed electron transfer and potassium uptake) do not proceed if ATP is replaced with AMP-PNP; however, the analog does affect these systems. Oxidative phosphorylation of whole mitochondria and submitochondrial preparations were unaffected by AMP-PNP.     

The metabolic effects and uptake of ethidium bromide by rat liver mitochondria.

The uptake of ethidium bromide by rat liver mitochondria and its effect on mitochondria, submitochondrial particles, and F₁ were studied. Ethidium bromide inhibited the State 4-State 3 transition with glutamate or succinate as substrates. With glutamate, ethidium bromide did not affect State 4 respiration, but with succinate it induced maximal release of respiration. These effects appear to depend on the uptake and concentration of the dye within the mitochondrion. In submitochondrial particles, the aerobic oxidation of NADH is much more sensitive to ethidium bromide than that of succinate. Ethidium bromide partially inhibited the ATPase activity of submitochondrial particles and of a soluble F₁ preparation. Ethidium bromide behaves as a lipophilic cation which is concentrated through an energy-dependent process within the mitochondria, producing its effects at different levels of mitochondrial function. The ability of mitochondria to concentrate ethidium bromide may be involved in the selectivity of the dye as a mitochondrial mutagen.     

Interaction of a synthetic polyanion with rat liver mitochondria

The effect of a polyanion (a copolymer of methacrylate, malaete and styrene in a 1:2:3 proportion with an average molecular weight of 10 000) on respiration, ATPase activity and ADP/ATP exchange activity of rat liver mitochondria and submitochondrial particles has been studied.The polyanion (at 17–150 μg/ml concentration, 100 μg polyanion corresponding to 0.83 μequiv. of carboxylic groups) inhibits the oxidation of succinate and NAD-linked substrates in state 3 in a concentration-dependent manner. The extent of this inhibition can be decreased by elevating the concentration of ADP. State 4 respiration is not affected by the polyanion. It has also a slight inhibitory effect on the oxidation of the above mentioned substrates in the uncoupled state (a maximum inhibition of 37% at 166 μg/ml polyanion concentration), which is unaffected by ADP. The strong inhibition of state 3 respiration can be relieved by 2,4-dinitrophenol to the low level observed in the uncoupled state. Ascorbate+TMPD oxidation is slightly inhibited in state 3, while it is not inhibited at all in the uncoupled state.The polyanion, depending on its concentration, strongly inhibits also the DNP-activated ATPase activity of mitochondria (50% inhibition at 40 μg/ml polyanion concentration).The ATPase activity of sonic submitochondrial particles is also inhibited. However, this inhibition is incomplete (reaching a maximum of 65%) and higher concentrations of the polyanion are required than to inhibit the ATPase activity of intact mitochondria.The polyanion inhibits the ADP/ATP translocator activity of mitochondria, measured by the “back exchange” of [2-³H]ADP. After a short preincubation of the mitochondria with the polyanion, the concentration dependence of the inhibition by the polyanion corresponds to that of the DNP-activated ATPase activity of intact mitochondria.It is concluded that, in intact mitochondria, the polyanion has at least a dual effect, i.e. it partially inhibits the respiratory chain between cytochrome b and cytochrome c, and strongly oxidative phosphorylation by blocking the ADP/ATP translocator.     

The use of aurovertin to determine the F1 content of submitochondrial particles and the ATPase complex

(1) The concentration of aurovertin-binding sites calculated from fluorimetric titrations of submitochondrial particles is equal to the F₁ concentration, calculated from the concentration of F₁-binding sites in stripped particles.(2) Direct binding experiments show that the fluorescence enhancement of aurovertin bound to submitochondrial particles and the isolated ATPase complex is less (or absent) at higher concentrations than at lower concentrations. The binding data can be described by ‘specific’ and ‘non-specific’ binding. The concentration of the ‘specific’ sites is twice that derived from fluorimetric titrations.(3) After dissociation of the bound F₁ with LiCl, fluorimetric titrations with aurovertin yield linear Scatchard plots. The fluorescence enhancement and K_D are equal to those of the β-subunit-aurovertin complex. The concentration of β-subunits is double the concentration of F₁.(4) It is concluded that both for submitochondrial particles and the isolated ATPase complex the most reliable and simple way to determine the F₁ content is to dissociate the F₁ with LiCl, spin down the insoluble material and titrate the supernatant (containing free β-subunit) with aurovertin.     

Interaction of aurovertin with submitochondrial particles,deficient in ATPase inhibitor

1. Binding of aurovertin to submitochondrial particles deficient in ATPase inhibitor is accompanied by an enhancement of the fluorescence by at least 100-fold.2. This change in fluorescence proceeds in three phases. The slowest change may be due to a conformational change in F₁, induced by the antibiotic bound during the rapid phases, giving rise to an increase in the quantum yield of the bound fluorochrome.3. Phosphate and ATP quench the fluorescence of the particle-aurovertin complex and ADP enhances it; the rate and extent of these changes are dependent on the availability of free Mg²⁺.4. There is at least one binding site on the submitochondrial particles, where ATP, ADP and phosphate can bind reversibly and for which these ligands compete. These interactions are dependent on the availability of free Mg²⁺ and are partly sensitive to oligomycin.5. Binding studies reveal two binding sites for aurovertin on inhibitor-free particles, one with high affinity and one with a lower affinity. Ligands such as phosphate and ATP decrease both the quantum yield and the affinity of the particles for aurovertin. They also increase the total concentration of binding sites, and affect the relative contribution of weak and strong binding sites.6. A model is presented in which changes of the aurovertin fluorescence reflect conformational changes of the ATPase induced by its ligands.     

Mitochondrial adenosine triphosphatase of the fission yeast, Schizosaccharomyces pombe 972h-. Changes in activity and inhibitor-sensitivity in response to catabolite repression.

1. The specific activity of mitochondrial ATPase (adenosine triphosphatase) in extracts of Schizosaccharomyces pombe decreased 2.5-fold as the glucose concentration in the growth medium decreased from 50mM to 15mM. 2. During the late exponential phase of growth, ATPase activity doubled. 3. Sensitivity to oligomycin and Dio-9 as measured by values for I50(mug of inhibitor/mg of protein giving 50% inhibition) at pH 6.8 increased sixfold and ninefold respectively during the initial decrease in ATPase activity, and this degree of sensitivity was maintained for the remainder of the growth cycle. 4. Increased sensitivity to NN'-dicyclohexylcarbodi-imide, triethyltin and venturicidin was also observed during the early stage of glucose de-repression. 5. Smaller increases in sensitivity to efrapeptin, aurovertin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diaz-le, quercetin and spegazzinine also occurred. 6. The ATPase of glycerol-grown cells was less sensitive to inhibitors than that of glucose-repressed cells; change in values for I50 were not so marked during the growth cycle of cells growing with glycerol. 7. When submitochondrial particles from glycerol-grown cells were tested by passage through Sephadex G-50, a fourfold increase in activity was accompanied by increased inhibitor resistance. 8. Gel filtration of submitochondrial particles from glucose-de-repressed cells gave similar results, whereas loss of ATPase occurred in submitochondrial particles from glucose-repressed cells. 9. It is proposed that alterations in sensitivity to inhibitors at different stages of glucose derepression may be partly controlled by a naturally occuring inhibitor of ATPase. 10. The inhibitors tested may be classififed into two groups on the basis of alterations of sensitivity of the ATPase during physiological modification: (a) oligomycin, Dio-9, NN'-dicyclohexylcarbodi-imide, venturicidin and triethyltin, and (b) efrapeptin, aurovertin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, quercetin and spegazzinine.     

ATP-dependent spectral response of oxonol VI in an ATP-Pi exchange complex

(1) Energy transduction in an ATPase complex (complex V) has been studied in two reactions catalyzed by this system, i.e., ATP-dependent spectral shift of oxonol VI, and ATP-P_i exchange activity. (2) Aurovertin alone inhibits 50% of the oxonol shift at 2 μM, and no further inhibition occurs at up to 12 μM. In combination with even weakly effective uncouplers, 4 μM aurovertin fully abolishes the oxonol response. No such effects are observed in the presence of oligomycin and uncouplers. (3) No pH gradient is detectable by quenching of 9-amino-6-chloro-2-methoxyacridine; and nigericin is without effect on the oxonol response. Valinomycin is inhibitory even in the absence of added potassium, due to ammonium ions introduced during the purification steps. Thiocyanate inhibits the dye response by only 10–27%, depending on the preparation. The extent of the oxonol response depends on the ATP / ADP ratio rather than the phosphorylation potential. (4) The dye response in the ATPase complex is 4–7-times less sensitive to bile salts than in submitochondrial particles. The inhibition by cardiolipin can be reversed by the addition of phospholipids. (5) The possibility is discussed that the oxonol response in the ATPase complex reflects, at least in part, a more local, ATP-dependent and energy-related process.     

Isolation and comparative studies of mitochondrial F1-ATPase from Morris hepatoma and rat liver.

A simple method of isolating mitochondrial ATPase from rat liver and Morris hepatoma cell lines by chloroform extraction and chromatography on DEAE-Sephadex is described. This method is suitable even when small amounts of starting material with relatively low specific ATPase activity (in the case of hepatoma mitochondria and submitochondrial particles) are available. The isolated enzyme from both rat liver and hepatomas had a high specific activity, was similarly activated by bicarbonate and 2,4-dinitrophenol, and had a typical five-band pattern in sodium dodecyl sulfate electrophoresis. Prior to DEAE-Sephadex chromatography, an additional protein band which migrates between the δ and ? subunits in the tumor F₁-ATPase preparation was observed. The purified enzymes were cold labile and restored oxidative phosphorylation function of F₁-ATPase depleted submitochondrial particles prepared from rat liver. The ATPase activity of the isolated enzymes was inhibited by mitochondrial ATPase inhibitor protein. The apparent stoichiometry of the inhibitor protein to the purified ATPase was extrapolated to be 2:1.     

Transplasma-membrane ferricyanide reduction in sycamore cells. Characterization of the system and inhibition by some phenyl biscarbamates

Evidence is presented for the presence of a transplasma-membrane redox system in sycamore cells, which were found able to reduce external ferricyanide. This reduction induced a simultaneous acidification of the external medium, with a H⁺/e⁻ stoichiometry of 0.925. Ferricyanide reduction was accompanied by a slight reduction of potassium uptake (15% inhibition). The transmembrane electron transport was inhibited by oligomycin and quinacrine; the effect of the latter inhibitor was only temporary, owing to its progressive absorption by the cells.At 100 μM, several derivatives of the herbicide phenmedipham were able to inhibit the growth of sycamore cells. These compounds inhibited the external acidification induced by fusicoccin, without interfering with the integrity of the plasmalemma. They had no effect on the phosphohydrolase activity of a microsomal fraction enriched in plasmalemma ATPase. They were not uncouplers of oxidative phosphorylation, and appeared as poor inhibitors of mitochondrial electron transfer (I₅₀ > 100 μM). In intact cell suspension, they inhibited both the reduction of ferricyanide and the accompanying acidification of the external medium (I₅₀ = 32 μM). Moreover, they could induce a reversal of the functioning of the redox system, which consequently induced ferrocyanide oxidation.     

The extent of mitochondrial F1-ATPase and adenine nucleotide carrier activity with ϵ-ATP

1. The use of 1,N⁶-ethenoadenosine 5′-triphosphate (?-ATP), a synthetic, fluorescent analog of ATP, by whole rat liver mitochondria and by submitochondrial particles produced via sonication has been studied.2. Direct [³H]adenine nucleotide uptake studies with isolated mitochondria, indicate the ?-[³H]ATP is not transported through the inner membrane by the adenine nucleotide carrier and is therefore not utilized by the 2,4-dinitrophenol-sensitive F₁-ATPase (EC 3.6.1.3) that functions in oxidative phosphorylation. However, ?-ATP is hydrolyzed by a Mg²⁺-dependent, 2,4-dinitrophenol-insensitive ATPase that is characteristic of damaged mitochondria.3. ?-ATP can be utilized quite well by the exposed F₁-ATPase of sonic submitochondrial particles. This ?-ATP hydrolysis activity is inhibited by oligomycin and stimulated by 2,4-dinitrophenol. The particle F₁-ATPase displays similar K_m values for both ATP and ?-ATP; however, the V with ATP is approximately six times greater than with ?-ATP.4. Since ?-ATP is a capable substrate for the submitochondrial particle F₁-ATPase, it is proposed that the fluorescent properties of this ATP analog might be employed to study the submitochondrial particle F₁-ATPase complex, and its response to various modifiers of oxidative phosphorylation.     

Kinetics of ATP synthesis in pea cotyledon submitochondrial particles

A kinetic study of oxidative phosphorylation by pea submitochondrial particles gave two K_m values for ADP, one low, the other high. The high value probably reflected a damaged site or a population of leaky mitochondria. Only the high affinity site with a low K_m for ADP was involved in ATP synthesis. α,β-Methylene ADP was found to be a competitive inhibitor of ATP synthesis. The inorganic phosphate analog, thiophosphate, decreased the apparent K_m of ADP while the rate of the reaction remained approximately the same. Adenyl imidodiphosphate, a specific inhibitor of ATP hydrolysis activity, had little effect on oxidative phosphorylation. A slight decrease in the K_m of the high affinity binding site for ADP was noted. Aurovertin was found to be a potent inhibitor of oxidative phosphorylation in pea submitochondrial particles. The K_m of the high affinity site was increased 10-fold. Also, the inhibition normally exerted by ADP on ATPase activity was severely reduced by aurovertin. In contrast, increasing the concentration of aurovertin only slightly affected the level of inhibition caused by adenyl imidodiphosphate on ATP hydrolysis.     

Assembly of a chimeric respiratory chain from bovine heart submitochondrial particles and cytochrome bd terminal oxidase of Escherichia coli

Cytochrome bd is a terminal quinol oxidase in Escherichia coli. Mitochondrial respiration is inhibited at cytochrome bc₁ (complex III) by myxothiazol. Mixing purified cytochrome bd oxidase with myxothiazol-inhibited bovine heart submitochondrial particles (SMP) restores up to 50% of the original rotenone-sensitive NADH oxidase and succinate oxidase activities in the absence of exogenous ubiquinone analogs. Complex III bypassed respiration and is saturated at amounts of added cytochrome bd similar to that of other natural respiratory components in SMP. The cytochrome bd tightly binds to the mitochondrial membrane and operates as an intrinsic component of the chimeric respiratory chain.     

Dicyclohexylcarbodiimide and vanadate sensitive ATPase of lung lamellar bodies

Lung surfactant is synthesized in lung epithelial type II cells and stored in the lamellar bodies prior to its secretion onto the alveolar surface. The lamellar bodies, like other secretory organelles, maintain an ATP-dependent pH gradient that is sensitive to inhibitors of H⁺-ATPase. This report shows that the ATPase activity of lamellar bodies is enriched in a fraction prepared from lamellar bodies that were disrupted after isolation. The apparent V_max for this enzyme was 150 nmol ATP hydrolyzed per min per mg protein and apparent K_m for ATP was approximately 50 μM. The enzyme activity was sensitive to N-ethylmaleimide (NEM), dicyclohexylcarbodiimide (DCCD) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-C1) (all inhibitors of vacuolar-type H⁺-ATPase) and vanadate (inhibitor of phosphoenzyme-type ATPase). Besides, the activity could also be inhibited with diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and Ca²⁺. Two proteins (of approximately 45 kDa and 17 kDa) of this fraction showed acid-stable phosphorylation with ATP. The labeling of proteins with ATP (-γ-³²P) could be chased with unlabelled ATP, suggesting that phosphorylation and dephosphorylation of these proteins is associated with the ATPase activity. Our results on inhibition characteristics of the enzyme activity suggest that besides a vacuolar type H⁺-ATPase, the lamellar bodies also contain a phosphoenzyme type ATPase that is sensitive to inhibitors of vacuolar type H⁺-ATPase.     

Effects of palmityl coenzyme A and palmitylcarnitine on phosphorylating respiration in heart mitochondria

Glutamate-supported respiration in mitochondria is inhibited by palmityl-CoA in the presence of carnitine. Palmityl-CoA-induced lag phase and depressed state 3 rates increase with increasing ADP. Palmityl-CoA inhibition of state 3 respiration with glutamate shows an increased I₅₀ for palmityl-CoA (three to fourfold) when ADP increases and carnitine is present. ADP alone has a small effect. Glutamate-supported respiration is more profoundly inhibited by palmityl-CoA (+carnitine) than palmityl-CoA oxidation. With palmityl-CoA (+ carnitine) alone, the I₅₀ for palmityl-CoA is two-to threefold greater than when glutamate is also present. Active respiration with palmityl-CoA as substrate demonstrates a 2.5-fold greater apparent affinity for ADP than when glutamate is also present. The kinetics are competitive in both cases. Palmitylcarnitine, above 30 μm, produces inhibition of glutamate-supported respiration, concomitant with mitochondrial swelling and eventual lysis. At 15 μm palmitylcarnitine (minimal swelling), succinate (+ rotenone)-supported respiration decreases with a decrease in K_app for ADP; no effect of 15–20 μm palmitylcarnitine on glutamate-supported respiration is observed. However, palmityl-CoA (+ carnitine)-inhibited respiration with glutamate is further decreased with 15 and 20 μm palmitylcarnitine, i.e., by 13 and 29%, respectively. Inhibition is competitive with ADP. With 3 μm palmitylCoA and 20 μm palmitylcarnitine, a decrease in carnitine (1.5 to 0.25 mm) decreases the apparent K_i for palmityl-CoA from 2.6 to 1.8 μm. The results suggest that glutamate increases the palmityl-CoA available to inhibit adenine nucleotide transport. Inhibition may take place external to the inner membrane. Competition of carnitine and palmitylcarnitine for substrate sites may explain the decreased apparent K_i for palmityl-CoA as carnitine decreases.     

Effects of respiratory chain inhibitors on mitochondrial ATPase activity

The effects of six respiratory chain inhibitors (amytal, rotenone, piericidin A, antimycin A, 2-N-heptyl-4-hydroxyquinoline N-oxide, and cyanide) on mitochondrial ATPase activity have been investigated. Each of these compounds inhibited the ATPase activity of intact mitochondria induced by uncoupling agents, ionophores, or alterations in ionic composition; the effects were variable depending upon the type and concentration of uncoupling agents or inhibitors utilized. The ATPase activity of sonicated submitochondrial particles was also diminished by respiratory inhibitors, but the isolated ATPase enzyme was not affected. We conclude from these results that these respiratory inhibitors interfere with the energy coupling mechanism of oxidative phosphorylation. The experimental observations tend to support the “chemical” theory, and appear to be less consistent with the “chemiosmotic” hypothesis of oxidative phosphorylation.     

A myxothiazol-sensitive Q-binding protein isolated from Chromatium vinosum

A quinol: ferricytochrome c oxidoreductase has been isolated from chromatophores of Chromatium vinosum by two procedures, involving extraction by bile salts and methanol, respectively. The steady-state kinetics indicate a random mechanism, with a K_m for 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinol of 1.1 μM and for the acceptor cytochrome c 1.75 μM. The enzyme is inhibited by myxothiazol, competitively with respect to quinol, with a K_i of about 2.3 μM. The protein reacts with ubiquinol produced by the succinate: Q oxidoreductase in submitochondrial particles or isolated succinate: cytochrome c reductase and can partially restore activity to myxothiazol-inhibited, antimycin-sensitive ubiquinol: cytochrome c oxidoreductase. The protein is considered to be analogous to the postulated myxothiazol-sensitive Q-binding protein in ubiquinol: cytochrome c oxidoreductase.     

The reconstitution of the mitochondrial energy-linked transhydrogenase.

Complex I (NADH-ubiquinone reductase) catalyzes pyridine nucleotide transhydrogenase at rates several fold higher than those found in submitochondrial particles from bovine heart. An ATP-dependent reduction of NADP⁺ by NADH was demonstrated after combination of Complex I with phospholipids, hydrophobic proteins derived from bovine heart mitochondria, and mitochondrial ATPase (F₁)¹. The reaction was inhibited by oligomycin, uncoupling agents and low concentrations of Triton X-100.