Kerstin Nordenbrand

[92a] Microsomal lipid peroxidation

Publisher Summary This chapter discusses the microsomal lipid peroxidation that may be demonstrated by measuring (1) O 2 consumption, (2) NADPH disappearance, and (3) malonaldehyde (MA) formation. Results of an experiment involving the measurement of all three parameters are illustrated. In the experimental procedure, rat liver microsomes are prepared s by sedimenting the 10,000 g supernatant of a 0.25M sucrose homogenate of rat liver at 105,000 g for 60 minutes. O 2 consumption is measured with a Clark O 2 electrode. NADPH disappearance is monitored fluorometrically at 450 mμ with an excitation wavelength of 365mμ. An Eppendorf photometer with fluorometer attachment is a suitable instrument for this purpose. MA formed is measured colorimetrically with the thiobarbituric acid (TBA) reaction. O 2 consumption may alternatively be measured manometrically in the Warburg apparatus. Measurement of NADPH disappearance spectrophotometrically at 340 mμ is complicated by the turbidity of the microsomal suspension which, in addition, may change during the incubation. The maximal rate of NADPH-linked lipid peroxidation at 30° is approximately 160 millimicromoles of O 2 consumed per minute per milligram of microsomal protein. The NADPH disappearance accompanying the microsomal lipid peroxidation ranges between one-third and one-fifth mole of NADPH per mole of O 2 consumed. The maximal extent of lipid peroxidation is approximately one micromole of O 2 consumed per milligram of microsomal protein.

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 (α‐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 UQHZ 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.

[14] Skeletal muscle mitochondria

Publisher Summary Bulk of the muscle tissue consists of myofibrils, and the mitochondria, as a rule, constitute a much smaller portion of the total mass of the tissue than in other organs. A crucial difference in the preparation of mitochondria from skeletal muscle as compared with other tissues is that sucrose, or in general, nonelectrolytes, cannot be used with advantage as the homogenizing medium. Like mitochondria from most animal tissues, skeletal muscle mitochondria contain the respiratory chain catalysts, cytochromes a, c, and b, ubiquinone, flavins, and pyridine nucleotides. The contents of the individual cytochromes and pyridine nucleotides and of ubiquinone in mitochondria from pigeon breast and rat leg muscle are presented in the chapter. Characteristic of skeletal muscle mitochondria is a high content of ubiquinone and a low content of NADP + . The respiratory activities of mitochondria from pigeon breast and human and rat skeletal muscle with various substrates are summarized. Skeletal muscle mitochondria also resemble mitochondria from other tissues in their capacity to catalyze the oxidation of various Krebs cycle metabolites. The chapter also discusses the assay for the respiration of isolated skeletal muscle mitochondria that can be measured by the conventional manometric or polarographie methods.

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.

Evidence for the occurrence in submitochondrial particles of a dual respiratory chain containing different forms of cytochrome b

Abstract 1. Addition of ascorbate + N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) to beef-heart submitochondrial particles (Mg-ATP particles) in the presence of KCN causes extensive reduction of cytochromes c1 + c and a + a3, and a partial reduction of cytochrome b. Addition of ATP results in an increased cytochrome b reduction, with a slight decrease in the level of reduced cytochrome a + a3. Subsequent addition of NAD+ causes a further substantial reduction of cytochrome b and a partial oxidation of cytochrome a + a3. The total amount of cytochrome b reduced at this stage is close to that obtained by adding NADH or succinate to the particles in the presence of KCN. 2. Cytochrome b reduced upon the addition of TMPD and NAD+ has an absorption maximum at 562 nm, and that reduced upon the addition of ATP at 565 nm, with a shoulder at 558 nm. All three phases of cytochrome b reduction are inhibited by antimycin, and the ATP- and NAD+-induced phases are also inhibited by FCCP and oligomycin. The NAD+-induced cytochrome b562 reduction is ATP-dependent, and is abolished by rotenone and by pyruvate + lactate dehydrogenase, indicating that it proceeds by way of NADH generated through reverse electron transport via cytrochrome b565 (+ b558). The effect of NAD+ in inducing cytochrome b562 reduction is duplicated by fumarate in a non-additive fashion. 3. The three phases of cytochrome b reduction are accompanied by roughly proportional extents of reduction of ubiquinone and flavorprotein. The amounts of cytochrome b, ubiquinone and flavoprotein, reduced upon the addition of ascorbate + TMPD, ATP and NAD+, are close to the total enzymically (NADH and succinate) reducible contents of these components in the particles. 4. The results are interpreted to indicate that submitochondrial particles contain a dual respiratory chain, one including cytochrome b565 (+ b558) and a functional Coupling Site II, and another, including cytochrome b562 and no functional Coupling Site II. The possible significance of these results for the functional organization of mitochondria is discussed.

Inhibition of purified mitochondrial ATPase (F1) by bathophenanthroline and relief of the inhibition by uncouplers

Abstract Low concentrations of bathophenanthroline inhibit the ATPase activity of purified beef-heart F 1 . The inhibition is antagonized by ATP in a fashion consistent with the involvement of a regulatory site on the enzyme. Various uncouplers, including FCCP, S-13, TTFB, dicoumarol and 2,4-dinitrophenol, relieve the bathophenanthroline inhibition, in concentrations similar to those known to uncouple mitochondrial oxidative phosphorylation.

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+.

[51] Mitochondrial ATPase inhibitor: Properties and applications

Publisher Summary Mitochondrial ATPase inhibitor has been isolated from beef heart, rat liver, and yeasts. Proteins with similar properties have been found in chloroplasts and bacteria as well as in myofibrils. Most procedures for the purification of the inhibitor take advantage of its high degree of stability to heat and to variations in pH. The inhibitor is highly sensitive to trypsin, a property that is commonly used to prepare inhibitor-free ATPase. Mitochondrial ATPase inhibitor lacks cysteine and tryptophan; ATPase inhibitors from spinach chloroplasts and Escherichia coli also lack tryptophan. The mitochondrial ATPase inhibitor is clearly distinct from the є subunit of F 1 based on amino acid composition and other properties. On the other hand, the chloroplast and E . coli inhibitors are thought to be identical with the є subunits of the corresponding ATPases. There is an extensive cross-reactivity between ATPase inhibitors and ATPases from various sources, including the myofibrillar troponin inhibitor (TNI) and actomyosin. However, this cross-reactivity does not seem to be general or even reciprocal. The inhibition of mitochondrial ATPase by mitochondrial ATPase inhibitor is noncompetitive or uncompetitive. A noncompetitive inhibition is also found in cross-reacting systems, for example, those consisting of TNI and ATPases from mitochondria or chloroplasts.