Effect of aflatoxin B 1 on respiration and oxidative phosphorylation in the rabbit. I. Studies of the hepatic mitochondria

The effect of various concentrations of aflatoxin B1 (AB1) is studied "in vitro" on rabbit liver mitochondria. AB1 inhibits at concentrations from 1 to 2-4 x 10(-4) M, the respiratory rate from 20% a maximum 41-35% by glutamate and succinate as substrates, but it does not uncouple oxidative phosphorylation nor does it inhibit site III (ascorbate + TMPD). The inhibited site appears to be between cytochromes b and c (c1). AB1 seems to be easily transported across the mitochondrial membrane. The maximum degree of inhibition and the promoting AB1 concentration are too high to explain the liver cell necrosis in rabbit induced by AB1 "in vivo".     

Interaction of n-alkanes with respiration and oxidative phosphorylation in rabbit heart mitochondria: n-nonane

Studies "in vitro" on the effect of n-nonane on coupled rabbit heart mitochondria with both succinate and glutamate as substrates show that the hydrocarbon examined makes the membrane permeable to protons (uncoupling), to some matrix enzymes and to exogenous NADH. The effect increases with increasing n-alkane concentration (from 0 to 160 microgram/mg mitochondrial protein) and temperature (from 15 degrees to 38 degrees C). Furthermore at higher concentrations and temperatures NADH oxidase inhibition is observed, whereas on succinate oxidase a biphasic effect (activation at lower concentrations and inhibition at higher concentrations) is produced. However the results, qualitatively similar to those observed with n-hexane, exhibit features probably due to a longer chain and that can be ascribed to perturbations of the physical state of membrane lipids.     

Interaction of n-alkanes with respiration and oxidative phosphorylation in rabbit heart mitochondria: n-hexane

n-Hexane on coupled rabbit heart mitochondria induces "in vitro" uncoupling with both glutamate and succinate as substrates and the effect increases with increasing n-alkane concentration (from 0 to 160 microgram/mg mitochondrial protein) and temperature (from 15 degrees to 38 degrees C). The inner mitochondrial membrane is made permeable to proteins; moreover extrusion of some matrix enzymes and entry of exogenous NADH is produced. Furthermore at higher concentrations and temperatures NADH oxidase inhibition and increase of its thermosensitivity is shown whereas upon succinate oxidase is evidenced a biphasic effect (activation followed by inhibition). The results, qualitatively similar to those observed with detergents and solvents, suggest a fluidization of the lipid phase of the membrane.     

Chemical modification of mitochondria. II. Effect of labeling on oxidative phosphorylation

The labeling of rat liver mitochondria (RLM) by the uncoupler 2,4-dinitro-5-(bromoacetoxyethoxy)phenol (DNBP) was studied and related to the effect of this molecule on oxidative phosphorylation. Alkylation of the cysteine residues was measured both with respect to incubation time of RLM with DNBP and with increasing DNBP concentration. At 3.3 × 10^?5m DNBP, the amount of S-carboxymethyl-cysteine formed was found to level off after about 3 min. The rate of ATP synthesis in RLM is reduced by increasing concentrations of DNBP and falls to zero, with either hydroxybutyrate or succinate as substrate, at 2 × 10^?4m DNBP. To characterize the effect of labeling on oxidative phosphorylation, the $\text{P}\text{O}$ ratio were measured after incubating RLM with DNBP for various times between 10 and 300 sec. The $\text{P}\text{O}$ ratio increases and tends to level off as the incubation time increases. No increase in $\text{P}\text{O}$ ratio was noted when RLM were similarly incubated with the nonlabeling uncoupler 2,4-dinitro-5-(acetoxyethoxy) phenol. Further, the effect of labeling on oxidative phosphorylation was determined with RLM which had been treated with DNBP and then washed free of the excess unreacted uncoupler. DNBP produces specific labeling in RLM which, when related to the effects of this uncoupler on oxidative phosphorylation, suggests that the labeled proteins may be involved in the primary energy transduction process.     

Effect of heliomycin on the respiration and oxidative phosphorylation of the liver mitochondria of the rat

The effect of heliomycin and known uncouplers of oxidative phosphorylation on respiration and oxidative phosphorylation was studied comparatively. Heliomycin, as well as 2,4-dinitrophenol, valinomycin and gramicidin S inhibited the mitochondrial synthesis of ATP. This process was inhibited completely by heliomycin at a concentration of 1.5 x 10(-5) M. The synthesis of inorganic pyrophosphate, the other macroergic compound, was also inhibited by heliomycin, ATPase and pyrophosphatase of uncoupled mitochondria being not inhibited by the antibiotic. Like 2,4-dinitrophenol, heliomycin stimulated the synthesis of ATPase and respiration in intact mitochondria. Probably, heliomycin inhibited the synthesis of ATP and pyrophosphate by uncoupling the processes of respiration and oxidative phosphorylation. It was shown earlier that heliomycin, a specific inhibitor of bacterial RNA synthesis, also affected energy metabolism of bacterial cells by inhibiting the synthesis of ATP and active transport.     

Inhibition of oxidative phosphorylation and respiration by ozone in tobacco mitochondria

Ozone was found to inhibit oxidative phosphorylation and oxygen uptake in mitochondria of tobacco leaves (Nicotiana tabacum, L. var. White Gold). The inhibition appeared to occur at both substrate and electron-transport chain levels. The inhibition increased with the length of exposure to ozone, however, the phosphorylative system was more sensitive to ozone than the respiratory system. With mitochondria from detached leaves after being treated with ozone at 1 ppm for 1 hour, uncoupling of phosphorylation was demonstrated without any detectable change in the rate of respiration in the early stage of ozone effect. Inhibition of phosphorylation by ozone was also demonstrated in isolated mitochondria without apparent change in optical density of the mitochondrial suspension at 520 mμ. Therefore, mitochondrial swelling appears not to be a necessary first step for ozone-induced uncoupling of phosphorylation. The evidence suggests that inhibition of oxidative phosphorylation in mitochondria may be a primary effect of ozone in tobacco leaves.

Sucrose and glucose, when fed to the detached tobacco leaves before ozone treatment, tended to raise the phosphorylative activity of mitochondria. Mannitol and lactose were less effective.

     

Effect of Fe2+- and gamma-induced lipid peroxidation on respiration and oxidative phosphorylation in the mitochondria

Lipid peroxidation activated by low concentrations of Fe2+ ions in a medium (1-5 microM) and gamma-quanta (10-50 Gy) stimulates the oxygen consumption and oxidative phosphorylation during the initial period of incubation (10-20 min). With relatively high concentrations of Fe2+ ions and higher radiation doses (50-100 Gy) inhibition of the activity of mitochondria is registered with respect to both indices.     

Interaction of n-alkanes with respiration and oxidative phosphorylation in rabbit heart mitochondria: n-dodecane, n-pentadecane and n-octadecane

The effect of n-dodecane, n-pentadecane and n-octadecane at various concentrations (from 0 to 160 microgram/mg mitochondrial protein) and temperatures (from 15 degrees to 38 degrees C) is studied "in vitro" on coupled rabbit heart mitochondria. With both glutamate and succinate as substrates, and at higher temperatures only, n-dodecane makes the membrane permeable to protons (uncoupling), to some matrix enzymes and poorly to NADH; moreover it slightly inhibits NADH-oxidase and exerts a biphasic action (activation followed by inhibition) on succinate oxidase. The results, qualitatively similar to those observed with n-hexane and n-nonane, are yet less striking and exhibit features probably due to the longer chain. The other n-alkanes examined do not show any effect.     

The effects of linoleate hydroperoxide on respiration and oxidative phosphorylation of rat liver mitochondria.

Linoleate hydropepoxide, purified by silica gel chromatography and at concentrations 70-100 nmol/mg mitochondrial protein, activated state 4 respiration and Mg-ATPase activity of mitochondria to levels of 80% and 25%, respectively, of those induced by 300 microM DNP, and completely inhibited oxidative phosphorylation. These effects are the same as those caused by linoleate, but the hydroperoxide caused more rapid degeneration of the activated respiration of mitochondria than linoleate. Further addition of the hydroperoxide induced oligomycin-insensitive Mg-ATPase to a level 3 times that obtained with DNP, accompanied by clearing of the mitochondrial suspension and release of malate dehydrogenase from the matrix. The extent of the effects caused by the methyl ester of linoleate hydroperoxide was much less than by the free acid.     

Energy-dependent accumulation of iron by isolated rat liver mitochondria V. Effect on factors controlling respiration and oxidative phosphorylation

1. Depending on the metabolic state, the addition of iron(III)-sucrose induces an inhibition or a stimulation of the respiration rate when added to isolated rat liver mitochondria.2. Under conditions identical to those used in the accumulation studies (Romslo, I. and Flatmark, T. (1973) Biochim. Biophys. Acta 305, 29?40), the ferric complex induces a decrease in the oxygen uptake concomitant to an oxidation of cytochromes c (+c₁) and a (+a₃). These results suggest that ferric iron is reduced to ferrous iron by the respiratory chain prior to or simultaneously with its energy-dependent accumulation.3. On the other hand, the addition of iron(III)-sucrose induces a stimulation of respiration in State 4 and State 3 provided Mg²⁺ is present in the suspending medium. In contrast to Ca²⁺, iron stimulates State 4 respiration in a cyclic process only within narrow concentration limits; at concentrations of iron above 100 μM the respiration remains in the activated state until anaerobiosis. The stimulation of State 4 respiration is more pronounced with succinate than with NAD-linked substrates, a difference which partly may be attributed to a stimulation of the succinate dehydrogenase complex.4. The stimulation of respiration by iron is approx. 3 times higher in State 3 than in State 4 and this difference can be attributed to a stimulation of the adenine nucleotide exchange reaction in State 3 with a concomitant increase in the rate of oxidative phosphorylation, although the $\text{P}\text{O}$ ratio is slightly diminished.