The mode of action of lipid-soluble antioxidants in biological membranes: relationship between the effects of ubiquinol and vitamin E as inhibitors of lipid peroxidation in submitochondrial particles.

The effects of ubiquinol and vitamin E on ascorbate- and ADP-Fe3+-induced lipid peroxidation were investigated by measuring oxygen consumption and malondialdehyde formation in beef heart submitochondrial particles. In the native particles, lipid peroxidation showed an initial lag phase, which was prolonged by increasing concentrations of ascorbate. Lipid peroxidation in these particles was almost completely inhibited by conditions leading to a reduction of endogenous ubiquinone, such as the addition of succinate or NADH in the presence of antimycin. Lyophilization of the particles followed by three or four consecutive extractions with pentane resulted in a complete removal of vitamin E and a virtually complete removal of ubiquinone, as revealed by reversed-phase high pressure liquid chromatography. In these particles, lipid peroxidation showed no significant lag phase and was not inhibited by either increasing concentrations of ascorbate or conditions leading to ubiquinone reduction. Treatment of the particles with a pentane solution of vitamin E (alpha-tocopherol) restored the lag phase and its prolongation by increasing ascorbate concentrations. Treatment of the extracted particles with pentane containing ubiquinone-10 resulted in a restoration of the inhibition of lipid peroxidation by succinate or NADH in the presence of antimycin, but not the initial lag phase or its prolongation by increasing concentrations of ascorbate. Malonate and rotenone, which prevent the reduction of ubiquinone by succinate and NADH, respectively, abolished, as expected, the inhibition of the initiation of lipid peroxidation in both native and ubiquinone-10-supplemented particles. Reincorporation of both vitamin E and ubiquinone-10 restored both effects.(ABSTRACT TRUNCATED AT 250 WORDS)

The mode of action of lipid-soluble antioxidants in biological membranes. Relationship between the effects of ubiquinol and vitamin E as inhibitors of lipid peroxidation in submitochondrial particles.

The effects of ubiquinol and vitamin E on ascorbate- and ADP-Fe(3+)-induced lipid peroxidation were investigated in beef heart submitochondrial particles before and after extraction of ubiquinone and vitamin E, and after reincorporation of either or both of these components. It is concluded that ubiquinol is capable of inhibiting lipid peroxidation without the mediation of vitamin E, but may also amplify the antioxidant effect of the latter. It is pointed out that ubiquinol is the only known lipid-soluble antioxidant that can be synthesized de novo in animal cells, and for which there exists an enzymic mechanism--the mitochondrial electron-transport system--that can regenerate the antioxidant from its oxidized form resulting from its inhibitory effect on lipid peroxidation. These features, together with its high degree of hydrophobicity and its general occurrence in biological membrane and in low-density lipoprotein, suggest a highly important role of ubiquinol in cellular defense against oxidative damage.

Inhibition of lipid peroxidation by ubiquinol in submitochondrial particles in the absence of vitamin E.

The relationship between the antioxidant effects of reduced coenzyme Q10 (ubiquinol, UQH2) and vitamin E (alpha-tocopherol) was investigated in beef heart submitochondrial particles in which lipid peroxidation was initiated by incubation with ascorbate + ADP-Fe3+. These effects were examined after extraction of coenzyme Q10 (UQ-10) and vitamin E from the particles and reincorporation of the same components alone or in combination. The results show that UQH2 efficiently inhibits lipid peroxidation even when vitamin E is absent. It is concluded that UQH2 can inhibit lipid peroxidation directly, without the mediation of vitamin E.

Microsomal lipid peroxidation: the role of NADPH--cytochrome P450 reductase and cytochrome P450.

The role of NADPH--cytochrome P450 reductase and cytochrome P450 in NADPH- and ADP--Fe3(+)-dependent lipid peroxidation was investigated by using the purified enzymes and liposomes prepared from either total rat-liver phospholipids or a mixture of bovine phosphatidyl choline and phosphatidyl ethanolamine (PC/PE liposomes). The results suggest that NADPH- and ADP--Fe3(+)-dependent lipid peroxidation involves both NADPH--cytochrome P450 reductase and cytochrome P450. Just as in the case of cytochrome P450-linked monooxygenations, the role of these enzymes in lipid peroxidation may be to provide two electrons for O2 reduction. The first electron is used for reduction of ADP--Fe3+ and subsequent addition of O2 to the perferryl radical (ADP--Fe3(+)-O2-), which then extracts an H atom from a polyunsaturated lipid (LH) giving rise to a free radical (LH.) that reacts with O2 yielding a peroxide free radical (LOO.). The second electron is then used to reduce LOO. to the lipid hydroperoxide (LOOH). In the latter capacity, reduced cytochrome P450 can be replaced by EDTA--Fe2+ or by the superoxide radical as generated through redox cycling of a quinone such as menadione.

Transport of Ca2+ and Mn2+ by mitochondria from rat liver, heart and brain.

The energy-dependent, respiration-supported uptake and the uncoupler- or Na+-induced release of Ca2+ and Mn2+ by mitochondria from rat liver, heart and brain were investigated, using as indicators radioisotopes (45Ca and 54Mn), proton ejection, oxygen consumption, nicotinamide nucleotide oxidation-reduction and, in the case of Ca2+, the metallochromic dye Arsenazo III. Ca2+ uptake in the presence of Pi was rapid in mitochondria from liver and brain, and less rapid in those from heart. Mn2+ uptake was much slower than that of Ca2+ in liver and heart, but only slightly slower in brain. When added together, Ca2+ accelerated the uptake of Mn2+, and Mn2+ retarded the uptake of Ca2+, by mitochondria from all three tissues. When Mn2+ was present during Ca2+ uptake, its own uptake remained accelerated even after Ca2+ uptake was terminated. Mg2+, which was not taken up, inhibited Ca2+ uptake by mitochondria from all three tissues, and, when present during Ca2+ uptake, accelerated the subsequent uptake of Mn2+. The uncoupler CCCP induced a release of both Ca2+ and Mn2+ from all three sources of mitochondria; yet, release of Mn2+ took place only in the absence of Pi. The release followed the same pattern as the uptake, i.e., Ca2+ accelerated the release of Mn2+ and Mn2+ retarded the release of Ca2+. Na+ induced a release of both Ca2+ and Mn2+ from heart and brain but not from liver mitochondria; again, Mn2+ release occurred only in the absence of Pi. The Na+-induced release of Ca2+ was inhibited by Mn2+, but the Na+-induced release of Mn2+ was not accelerated by Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Mn2+ prevents the Ca2+-induced inhibition of ATP synthesis in brain mitochondria.

Uptake of Ca2+ by rat brain mitochondria causes an inhibition of respiratory stimulation by ADP, and the inhibition is relieved upon Na+-induced release of Ca2+ from the mitochondria, in accordance with earlier reports. We show that simultaneous uptake of Ca2+ and Mn2+ results in no inhibition of ADP-stimulated respiration, indicating that Mn2+ prevents the Ca2+-induced inhibition of ATP synthesis, without preventing Ca2+ accumulation in the mitochondria. The results are discussed in relation to a possible involvement of the mitochondrial ATPase-inhibitor protein in the observed effects of Ca2+ and Mn2+.

The interaction of mitochondrial F1-ATPase with the natural ATPase inhibitor protein.

The interaction of soluble mitochondrial ATPase from beef heart with the natural ATPase inhibitor was studied. It was found that the phosphorylation of small amounts of ADP by phosphoenolpyruvate and pyruvate kinase, and an ensuing catalytic cycle supports the binding of the inhibitor to the enzyme. The association of the inhibitor with F1-ATPase does not increase the content of ATP in the F1-ATPase-inhibitor complex. The inhibitor of catalytic activity bathophenanthroline-Fe2+ chelate prevents the interaction, while the association of the inhibitor with F1-ATPase is delayed if the reaction is carried out in 2H2O. The date indicate that a transient state involved in the catalytic cycle is the form of the enzyme that interacts with the inhibitor. The proton-motive force-induced dissociation of the inhibitor from particulate ATPase is prevented by bathophenanthroline-Fe2+ chelate and nitrobenzofurazan chloride, which indicates that a functional catalytic (beta) subunit is required for the proton-motive force-induced release of the inhibitor. The data suggest a direct involvement of catalytic (beta) subunit in the mechanism by which the F1-ATPase senses the proton-motive force.