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PubMed1965

On the mechanism of respiratory control.

Control of oxidation is the key mechanism in the regulation of energy metabolism. In glycolysis the oxidation of glyceraldehyde-3-phosphate is controlled by DPNH, which inhibits glyceraldehyde-3-phosphate dehydrogenase. In oxidative phosphorylation the inhibition of electron flow from DPNH to oxygen, called "respiratory control," is the subject of this paper. After a discussion of the physiological significance of the "tight coupling" between phosphorylation and oxidation, studies on "loosely coupled" submitochondrial particles are reported. These particles are capable of oxidative phosphorylation in the presence of a suitable phosphate acceptor system, but in contrast to controlled, intact mitochondria they oxidize DPNH in the absence of phosphate and ADP. The addition of o-phenanthroline to submitochondrial particles gives rise to an inhibition of respiration, which is partly reversed by phosphate and ADP or by dinitrophenol. The properties of this model system of respiratory control will be described.

Adenine Nucleotides↗
PubMed1982

Spontaneous aggregation of the mitochondrial natural ATPase inhibitor in salt solutions as demonstrated by gel filtration and neutron scattering. Application to the concomitant purification of the ATPase inhibitor and F1-ATPase.

(1) The natural ATPase inhibitor (IF1) from beef heart mitochondria has a tendency to form aggregates in aqueous solutions. The extent of aggregation and the structure of the aggregates were assessed by gel filtration and small-angle neutron scattering. IF1 polymerization was found to depend on the salt concentrations, pH of the medium and concentration of IF1. The higher the salt concentration, the lower the aggregation state. Aggregation of IF1 was decreased at slightly acidic pH. It increased with the concentration of IF1 as expected from the law of mass action. (2) Neutron scattering showed the aggregation of IF1 in 2 M ammonium sulfate solutions. The predominant species is the dimer which has a somewhat elongated shape. (3) The Sephadex G-50 chromatography that is supposed to deprive beef heart submitochondrial particles of loosely bound IF1 (Racker, E. and Horstman, L.L. (1967) J. Biol. Chem. 242, 2547-2551) was shown to have a limited effectiveness as a trap for IF1. The reason was that IF1 released from the particles formed high molecular weight aggregates that were not separated from the membrane vesicles by Sephadex G-50 chromatography. (4) The above observations provide the basis for a simple method of purification of beef heart IF1 which combines the recovery of the supernatant from submitochondrial particles with the last three steps of the IF1 preparation described by Horstman and Racker (J. Biol. Chem. (1970) 265, 1336-1344). The particles recovered in the sediment were deprived of IF1 and could therefore be used for preparation of F1-ATPase. The advantage of this method is that both IF1 and F1-ATPase can be prepared from the same batch of mitochondria.

Adenosine Triphosphatases↗
PubMed1981

The presence of two hydrolytic sites on beef heart mitochondrial adenosine triphosphatase.

The ribose-modified nucleotides 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) and TNP-ADP were used to probe the catalytic sites on soluble beef heart mitochondrial adenosine triphosphatase (F1). Both compounds were potent competitive inhibitors of ATP hydrolysis catalyzed by F1, Ki = 5.5 and 10 nM, respectively, and by submitochondrial particles, Ki (TNP-ATP) = 21 nM. Both compounds also were potent competitive inhibitors of ATP synthesis during oxidative phosphorylation (Ki = 1300 nM). Both analogs inhibited the 32Pi-ATP exchange reaction and the ATP-dependent reduction of NAD+ by succinate, catalyzed by submitochondrial particles. TNP-ATP and TNP-ADP were bound by F1. The presence of two binding sites on the enzyme for TNP-adenine nucleotides was determined by titrations of difference absorbance spectra, of the increase in fluorescence of the analog which occurred upon interaction with protein, and by titrations with the centrifuge column method using 32P-labeled TNP-adenine nucleotides. The first binding site bound the analogs with an affinity too high to be measured. The Kd for analog binding by the second site was 20 to 80 nM. In the presence of Mg2+, the 2 sites were filled with the TNP-ATP at a rate too rapid to be resolved by the procedure used. TNP-[gamma-32P]ATP was hydrolyzed by F1, Km = 0.2 microM, Vmax = 1.1 mol of 32Pi formed/mol of F1/s. It was shown, using the isotope trap technique as well as the inhibitor efrapeptin, that the 2 binding sites for TNP-ATP on F1 are hydrolytic sites.

Adenine Nucleotides↗
PubMed1981

Interaction between ubiquinone and ATPase in mitochondrial membranes.

The extraction of ubiquinone from mitochondrial membranes produces alterations of ATPase activity including a reversible loss of oligomycin sensitivity which is restored by long-chain Q-homologs, Short-chain ubiquinones like Q3 produce a loss of oligomycin and dicyclohexyl carbodiimide (DCCD) sensitivity in submitochondrial particles. The effect shows uncompetitive or noncompetitive Kinetics with respect to oligomycin or DCCD respectively. Long-chain ubiquinones have a competitive effect with Q3, thus restoring oligomycin sensitivity; they behave, however, in about the same way as Q3 in lowering the DCCD sensitivity in submitochondrial particles. On the basis of these observations we suggest that ubiquinone may be a physiological modulator of ATPase activity in the mitochondrial membrane.

Adenosine Triphosphatases↗
PubMed1998

Production of nitric oxide by mitochondria.

The production of NO. by mitochondria was investigated by electron paramagnetic resonance using the spin-trapping technique, and by the oxidation of oxymyoglobin. Percoll-purified rat liver mitochondria exhibited a negligible contamination with other subcellular fractions (1-4%) and high degree of functionality (respiratory control ratio = 5-6). Toluene-permeabilized mitochondria, mitochondrial homogenates, and a crude preparation of nitric oxide synthase (NOS) incubated with the spin trap N-methyl-D-glucamine-dithiocarbamate-FeII produced a signal ascribed to the NO. spin adduct (g = 2.04; aN = 12.5 G). The intensity of the signal increased with time, protein concentration, and L-Arg, and decreased with the addition of the NOS inhibitor NG-monomethyl-L-arginine. Intact mitochondria, mitochondrial homogenates, and submitochondrial particles produced NO. (followed by the oxidation of oxymyoglobin) at rates of 1.4, 4.9, and 7.1 nmol NO. x (min.mg protein)-1, respectively, with a Km for L-Arg of 5-7 microM. Comparison of the rates of NO. production obtained with homogenates and submitochondrial particles indicated that most of the enzymatic activity was localized in the mitochondrial inner membrane. This study demonstrates that mitochondria are a source of NO., the production of which may effect energy metabolism, O2 consumption, and O2 free radical formation.

Animals↗
PubMed1999

Fragility of the electron transport chain and superoxide generation in mitochondria of the liver graft after cold ischemia.

BACKGROUND: After cold ischemia, electrons transferred in the electron transport chain may leak out of the mitochondria in proportion to the deterioration of mitochondrial oxidative phosphorylation. This seems to be one major cause of the lipid peroxidation that occurs mainly in the hepatocytes at reperfusion in liver transplantation. To examine this hypothesis, we investigated superoxide generation and the amount of oxidative phosphorylation in the mitochondria isolated from rat livers after cold preservation. METHODS: Rat liver was preserved in University of Wisconsin solution at 4 degrees C for 24 hr. The mitochondrial fraction was prepared, and the amount of ATP synthesis and superoxide generation was investigated. Superoxide generation in the electron transport chain of submitochondrial particles was also measured by a chemiluminescence recorder. RESULTS: The amount of ATP synthesis was significantly decreased after 12 hr of cold preservation. In the whole mitochondria, superoxide production in the presence of succinate was approximately 1/2000 to 1/3000 less than that observed in the submitochondrial particles at any determination point, and superoxide production was not affected by cold preservation. In the presence of antimycin A, superoxide production in the mitochondria after 18 hr of preservation increased significantly. CONCLUSION: These results indicate that the electron transfer in the complex III of the mitochondrial membrane becomes leaky after long periods of cold ischemia, but that leakage of superoxide anion did not increase, although the mitochondrial respiratory phosphorylation was deteriorated. We conclude that superoxide through the mitochondrial membrane cannot cause lipid peroxidation in hepatocytes at reperfusion even after a long period of cold ischemia.

Adenosine Triphosphate↗
PubMed1994

Inhibition by nilutamide of the mitochondrial respiratory chain and ATP formation. Possible contribution to the adverse effects of this antiandrogen.

The effects of nilutamide on the mitochondrial respiratory chain were investigated in rats. In isolated mitochondria, nilutamide (100 microM) inhibited respiration that was supported by substrates feeding electrons into complex I of the respiratory chain but did not inhibit respiration that was supported by substrates donating electrons to complexes II, III or IV. Inhibition of complex I occurred without any lag time. In submitochondrial particles, nilutamide (100 microM) decreased both oxygen consumption mediated by NADH and the oxidation of NADH; addition of superoxide dismutase and catalase did not alleviate inhibition. There was no electron spin resonance evidence for detectable mitochondrial formation of the nilutamide nitro anion free radical by submitochondrial particles or for the formation of iron-nitrosyl complexes with mitochondrial Fe-S clusters in isolated hepatocytes. Severe inhibition of complex I by nilutamide (500 microM) led to upstream inhibition of fatty acid beta-oxidation. Nilutamide (100 microM) decreased the mitochondrial membrane potential and ATP formation in mitochondria energized by malate plus glutamate. In hepatocytes incubated without glucose, nilutamide (500 microM) led to an early (2 hr) drop in cellular ATP and early (4 hr) toxicity. With 5 mM glucose, however, ATP was not decreased and toxicity was mild at these early times. It was concluded that nilutamide itself inhibited the mitochondrial respiratory chain at the level of complex I and decreased ATP in hepatocytes incubated without glucose, which resulted in early toxicity. In the presence of glucose, ATP was not depleted at early times and delayed toxicity was probably the result of an oxidative stress (as previously reported).

Adenosine Triphosphate↗
PubMed1976

Reconstitution and characterization of the adenine nucleotide transporter derived from bovine heart mitochondria.

1. Adenine nucleotide exchange-transport was reconstituted in vesicles prepared from phospholipids and protein fractions derived from bovine heart submitochondrial particles. The transport, which was specific for ATP and ADP was measured either as ADP/ADP, ATP/ATP, or ADP/ATP exchange. The highest specific activity (370 nanomoles of ADP/ADP exchange/min/mg of protein at room temperature) was obtained with a protein fraction prepared by cholate extraction of partly resolved submitochondrial particles followed by ammonium sulfate fractionation. 2. At 200 muM external nucleotide, the exchange reactions were inhibited by low concentrations of bongkrekate, atractyloside, and palmitoyl-CoA, with Ki values of 1.8, 3.0, and 7.5 muM, respectively. The ADP/ADP nucleotide exchange was stimulated about 5-fold by 500 muM MgCl2 or MnCl2(km of 40 muM) and about 3-fold by 500 muM CaCl2(Km of 90 muM). It was optimal between pH 6.0 and 7.0 and decreased rapidly above pH 7.5. Arrhenius plots between 0 degrees and 40 degrees showed a break point at 15 degrees with soybean phospholipids and an activation energy of 29.5 kcal/mole from 0 degrees-15 degrees and 9.0 kcal/mole from 15 degrees-40 degrees. With mitochondrial phospholipids the break point was at 9 degrees and activation energies were 42.4 kcal/mole from 0 degrees-9 degrees and 7.6 kcal/mole from 9 degrees-40 degrees. 3. The phospholipid requirements for adenine nucleotide exchange were similar to those of oxidative phosphorylation. Optimal rates were observed with a phosphatidylethanolamine to phosphatidylcholine ratio of 4:1. Cardiolipin had a slight stimulatory effect. 4. The uptake of ADP into vesicles containing ATP was stimulated by KCl or by KPi as well as by hexafluoracetonylacetone, and uncoupler of oxidative phosphorylation. The uptake of ATP into vesicles containing ADP was inhibited by KCl or by KPi, but was also stimulated by hexafluoracetonylacetone. In both cases valinomycin reversed the effects of KCl, while mersalyl or N-ethylmaleimide prevented the effects of KPi. In contrast, none of these salts nor hexafluoracetonylactone affected the ADP/ADP or ATP/ATP exchange. These findings suggest that in the reconstituted system the ADP/ATP exchange is electrogenic.

Animals↗
PubMed1984

Novel isolation of ubiquinone-binding proteins located in different sites of beef heart mitochondrial respiratory chain.

Ubiquinone-binding proteins were isolated from beef heart submitochondrial particles by a simple fractionation procedure using ethanol and ammonium acetate after solubilization of the particles with deoxycholate. The ubiquinone-binding proteins were further purified by passing them through a column of phenyl-Sepharose CL-4B to eliminate the free form of ubiquinone and hydrophobic proteins such as cytochromes. At least 25% of the total ubiquinone present in the submitochondrial particles was associated with the purified proteins. Applying the same method, ubiquinone-binding proteins could be isolated from complexes I and III.

Animals↗
PubMed1989

[Kinetics of the interaction of ATPase of submitochondrial fragments and a natural protein-inhibitor].

Conditions were selected which enable a quantitative assay of the ATPase inhibitor protein in submitochondrial particles. It was found that the isolated soluble inhibitor exhibits a marked pH-dependent hysteretic behaviour, i. e., an instant jump of pH for the inhibitor solution from 4.8 to 8.2 induced a slow alteration of its activity as measured by the inhibition of ATP hydrolysis by submitochondrial particles. In acid media (pH less than 6.8), the inhibitor is in the active, whereas in alkaline media (pH greater than 6.8) in the inactive state; the apparent pKa value for the cooperative active/inactive transition is 6.8. Treatment of the inhibitor protein with diethylpyrocarbonate, a specific reagent for histidine, completely abolishes its inhibitory activity. Two types of the inhibitor protein--ATPase interaction were revealed, i.e., reversible (ATP-independent) and irreversible (ATP-dependent) ones. Both reactions, i.e., ATP hydrolysis and ATP inhibition by the inhibitor in the presence of Mg2+ are characterized by a hyperbolic dependence of the reaction rate on ATP concentration; however, for both reactions the apparent KmATP values (50 and 5 microM, respectively) differ significantly (pH 8.0). Thus, the inhibitor--ATPase interaction shows that there exists a specific site for ATP in the ATPase which is different from the catalytic one. A model for the inhibitor protein interaction with ATPase which takes account of a slow pH-dependent conformational transformation of the inhibitor protein is proposed.

Adenosine Triphosphatases↗
PubMed1986

Anomalous oxygen-18 exchange during ATP synthesis in oxidative phosphorylation.

The synthesis of ATP from highly enriched [18O]Pi by submitochondrial particles driven by succinate oxidation produces distributions of 18O-labeled ATP species that deviate from the distributions predicted by a simple model for the exchange. Control experiments indicate no change in isotopic distribution when [18O]ATP is synthesized from [18O]ADP by adenylate kinase, which is bound to the submitochondrial particles. The observed deviations are in the opposite direction from that produced by heterogeneity due to multiple pathways for ATP synthesis. Two types of complex models can account for the observed deviations. One model has nonequivalence of the Pi oxygens during the exchange reaction, due to incomplete randomization of the Pi oxygens during the reversible cycles of hydrolysis and synthesis of bound ATP. The other model assumes that, during each turnover, a slow transition must occur between a high-exchange and a low-exchange pathway.

Adenosine Triphosphate↗
PubMed1990

Inhibition by capsaicin of NADH-quinone oxidoreductases is correlated with the presence of energy-coupling site 1 in various organisms.

The NADH-ubiquinone reductase activity of the respiratory chains of several organisms was inhibited by capsaicin and dihydrocapsaicin, which are the pungent principles of red pepper. This inhibition was correlated with the presence of an energy transducing site in this segment of the respiratory chain. Where the NADH-quinone oxidoreductase segment involved an energy coupling site (e.g., in Paracoccus denitrificans, Escherichia coli, and Thermus thermophilus HB-8 membranes and bovine heart mitochondria), capsaicin acted as an inhibitor of ubiquinone reduction by NADH. In contrast, where this energy coupling site was absent (e.g., in Saccharomyces cerevisiae mitochondria and Bacillus subtilis membranes), there was no inhibition of NADH-ubiquinone reductase activity by capsaicin. The capsaicin inhibition of Paracoccus membranes was reversed by washing the membranes with medium containing bovine serum albumin. In the E. coli and Paracoccus membranes and bovine submitochondrial particles, capsaicin acted as a noncompetitive inhibitor for ubiquinone-1 at lower concentrations of ubiquinone-1 (less than 20 microM) and as a competitive inhibitor at higher concentrations of ubiquinone-1 (greater than 50 microM). In addition, the concentrations of capsaicin required for 50% inhibition of NADH oxidase activity of bovine submitochondrial particles were increased when ubiquinone-10 was added to the particles. The mechanism by which capsaicin inhibits the energy-transducing NADH-quinone oxidoreductase is discussed.

Animals↗
PubMed1975

Physical and enzymatic properties of nucleotide-depleted beef heart mitochondrial adenosine triphosphatase.

Tightly bound adenine nucleotides are removed from multiple binding sites on beef heart mitochondrial ATPase (F1) by chromatography on columns of Sephadex equilibrated with 50% glycerol. Release of nucleotides from the enzyme is associated with large decreases in sedimentation velocity (from 11.9 S to 8.4 S) which may be observed in concentrated solutions of polyols. Polyol-induced conformational changes are reversed when the enzyme is returned to dilute buffers. The nucleotide-depleted enzyme restores oxidative phosphorylation in F1-deficient submitochondrial particles. Reconstitution of nucleotide-depleted F1 with the ATP analog (adenylyl-imidodiphosphate (AMP-PNP), almost 5 moles of AMP-PNP per mole of enzyme, results in preparations with substantially inhibited ATPase activity which nevertheless restores oxidative phosphorylation and the 32Pi-ATP exchange reaction in F1-deficient submitochondrial particles. Incubation of the analog-labeled enzyme with ATP and Mg++ results in partial displacement of the analog and a time-dependent recovery of ATPase activity.

Adenine Nucleotides↗
PubMed1995

Inhibitory effect of the antioxidant ethoxyquin on electron transport in the mitochondrial respiratory chain.

Ethoxyquin (6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline, EQ) is an antioxidant used as a preservative in animal and human foods. In a previous work, we demonstrated that EQ induces an inhibition of renal secretory mechanisms that are dependent on metabolic energy; EQ inhibits renal ATPases. In the present study, we analyzed the effects of EQ on the metabolic pathways of renal and hepatic rat cells, as well as on mitochondrial and submitochondrial particles isolated from bovine heart and kidney. EQ induced a mild inhibition of oxygen uptake when it was added to whole homogenates of rat renal cortex in the presence of glucose. In contrast, a strong concentration-dependent inhibition was produced when EQ was added to preparations of intact liver mitochondria or to submitochondrial particles isolated from renal cortex. In the presence of NADH, 90% inhibition was attained at a final concentration of 1 mM EQ. The direct inhibitory effect of EQ on NADH dehydrogenase was a most relevant finding, since no inhibitor for the partial reaction of NADH-ferricyanide on this complex has been reported previously.

Animals↗
PubMed1994

Age dependent changes in mitochondrial FoF1 ATP synthase in regenerating rat-liver.

The effect of aging on rat liver regeneration and on the FoF1-ATP synthase complex of isolated liver mitochondria was followed after partial (70%) hepatectomy. ATP hydrolase activity in submitochondrial particles prepared from regenerating liver was first depressed; the time needed to reach the lowest activity was age dependent. This decrease was accompanied by parallel decrease of i) the respiratory rate of succinate supplemented mitochondria in state III; ii) the respiratory control index; iii) the rate of synthesis of ATP in succinate supplemented submitochondrial particles. This first phase of liver regeneration, characterized at all ages by a lag phase in the growth, was followed by a second phase in which the tissue mass was restored and the enzyme activities normalized. Immunoblot analysis showed that the changes in the catalytic activities of the FoF1-ATP synthase observed during liver regeneration were accompanied by parallel changes in the amount of subunits of both the catalytic (F1) and the membrane (Fo) sector of the complex.

Aging↗
PubMed1975

Symmetry, orientation and rotational mobility in the a3 heme of cytochrome c oxidase in the inner membrane of mitochondria.

The photoinduced linear dichroism of absorption changes resulting from photolysis of the complex between heme a3 of the cytochrome oxidase and CO is studied. The experiments started from isotropic solutions or suspensions of the enzyme both in its isolated form and in mitochondria. The anisotropy responsible for the linear dichroism was induced by excitation with a flash of linearly polarized light. The dichroic ratios observed with various systems; polymerized enzyme in solution, enzyme in mitochondria and in submitochondrial particles (at 20 degrees C as well as at liquid N2-temperature) all approached a value of 4/3 which characterizes a chromophore which is circularly degenerate. Therefrom we conclude that the interaction of heme a3 with its microenvironment within the protein does not break its four-fold symmetry. The experiments with mitochondria and submitochondrial particles suspended in aqueous buffer revealed similarly high dichoric ratios without any dichroic relaxation other than a rather slow one which could be attributed to the rotation of the whole organelle in the suspending medium. Therefrom we conclude that the cytochrome oxidase either is totally immobilized in the membrane, or that it carries out only limited rotational diffusion around a single axis coinciding with the symmetry axis of heme a3. In the light of independent evidence for a transmembrane arrangement of the oxidase and for the general fluidity of the inner mitochondrial membrane we consider anisotropic mobility of the cytochrome oxidase around an axis normal to the plane of the membrane as the most likely interpretation. Then our experimental results imply that the plane of heme a3 is coplanar to the membrane.

Animals↗
PubMed1989

Structure-function relationships of mitochondrial ATPase-ATPsynthase using Schizosaccharomyces pombe yeast mutants with altered F1 subunits.

Phenotypic revertants have been selected from mutants of the yeast Schizosaccharomyces pombe devoid of either alpha or beta subunits of mitochondrial ATPase-ATPsynthase. In contrast to parental mutants, phenotypic revertants are able to grow on glycerol respiratory medium and show immunodetectable alpha and beta subunits. However, growth and cellular respiration are only partially restored as compared to the wild strain, indicating that the recovered subunits are mutated. ATPase activity of revertant submitochondrial particles shows markedly different parameters: more acidic optimal pH, absence of bicarbonate activation and decreased sensitivity to azide inhibition in the alpha subunit-modified R3.51. Opposite differences are observed in the beta subunit-modified R4.3: more alkaline optimal pH, much higher bicarbonate activation, and increased sensitivity to azide. The ITPase activity of R4.3 submitochondrial particles is also more sensitive to azide as compared to the wild strain. ATPase activity of purified F1 also exhibits marked differences: loss of bicarbonate-sensitive negative cooperativity, decreased sensitivity to both ADP and azide inhibitions in the R3.51 revertant. On the contrary, increased negative cooperativity and increased sensitivity to both ADP and azide inhibitions are observed for the R4.3 revertant enzyme which in addition exhibits a much lower maximal rate. The beta subunit-mutation of R4.3 also increases the sensitivity of ITPase activity to tripolyphosphate inhibition, whereas the alpha subunit-mutation of R3.51 is without any effect. Soluble F1 with beta subunit-mutation is very sensitive to high ammonium sulfate concentrations required for enzyme precipitation and concentration and known to partially deplete the enzyme from its endogenous nucleotides. On the contrary, poly(ethylene)glycol is very efficient for preparing from any strain a pure and very stable enzyme retain-ing high amounts of endogenous nucleotides. The R4.3 revertant F1 retains even more nucleotides than the wild-strain F1 and is much less sensitive to high iodide concentrations which favor enzyme dissociation and precipitation. The tryptophan intrinsic fluorescence of F1 is modified by both mutations that increase the maximal emission intensity. The most important effect is produced by beta subunit-mutation which decreases the quenchable fraction, one-third to one-half tryptophans being no longer accessible to iodide. The overall results suggest that both mutations modify enzyme-nucleotide interactions: the alpha subunit-mutation of R3.51 would favor ADP release by lowering interactions with the adenine moiety, whereas the beta subunit-mutation of R4.3 would lower ADP release by strengthening interactions with the phosphate chain moiety.

Adenosine Triphosphatases↗
PubMed1990

The photodynamic effect of rose bengal on proteins of the mitochondrial inner membrane.

Photodynamic action promoted by Rose Bengal was evaluated in solutions of unsaturated fatty acids or histidine, and on beef heart submitochondrial particles. Rose Bengal-promoted photooxidation of histidine was mainly due to the opening up of the imidazole ring by singlet oxygen. Photosensitization of polyunsaturated fatty acids (PUFA) resulted in oxygen consumption and thiobarbituric acid-reactive substances (TBARS) formation, the extent of which was linearly related to the increasing degree of unsaturation. Photosensitization of submitochondrial particles caused oxygen consumption and TBARS production. These processes involved two different reaction components: during the first, most of the mitochondrial proteins were inactivated, the most sensitive being succinate dehydrogenase and cytochrome c. The values for the rate ratios of [TBARS] formation/[O2] consumption for the first and second phase were 0.36 and 1.32%, respectively, pointing to a larger contribution of lipid peroxidation during the second phase. The calculation of the rate constants for reaction of singlet oxygen with mitochondrial proteins suggests that singlet oxygen is more reactive towards proteins than to PUFA. The biological role of this selectivity is discussed in terms of the mitochondria as one of the first targets for photosensitized reactions.

Animals↗