Succinate cytochrome c reductase, activity

Abnormalities of the respiratoiy chain. These are increasingly identified as the hallmark of mitochondrial diseases or mitochondrial encephalomyopathies [13]. They can be identified on the basis of polarographic studies showing differential impairment in the ability of isolated intact mitochondria to use different substrates. For example, defective respiration with NAD-dependent substrates, such as pyruvate and malate, but normal respiration with FAD-dependent substrates, such as succinate, suggests an isolated defect of complex I (Fig. 42-3). However, defective respiration with both types of substrates in the presence of normal cytochrome c oxidase activity, also termed complex IV, localizes the lesions to complex III (Fig. 42-3). Because frozen muscle is much more commonly available than fresh tissue, electron transport is usually measured through discrete portions of the respiratory chain. Thus, isolated defects of NADH-cytochrome c reductase, or NADH-coenzyme Q (CoQ) reductase suggest a problem within complex I, while a simultaneous defect of NADH and succinate-cytochrome c reductase activities points to a biochemical error in complex III (Fig. 42-3). Isolated defects of complex III can be confirmed by measuring reduced CoQ-cytochrome c reductase activity. 

Defects of complex II. These have not been fully characterized in the few reported patients, and the diagnosis has often been based solely on a decrease of succinate-cytochrome c reductase activity (Fig. 42-3). However, partial complex II deficiency was documented in muscle and cultured fibroblasts from two sisters with clinical and neuroradiological evidence of Leigh s syndrome, and molecular genetic analysis showed that both patients were homozygous for a point mutation in the flavoprotein subunit of the complex [17]. This was the first documentation of a molecular defect in the nuclear genome associated with a respiratory chain disorder. 

The insect fat body is considered to be the functional analog of the vertebrate liver, and it is of interest, therefore, to note that mitochondrial fractions from the fat body of the cockroach Blaherus discoidalis reveal increases in enzymic activity during development similar to those reported for mammalian liver (Keeley, 1972). Cytochrome oxidase and succinate-cytochrome c reductase activities do not increase in parallel. Immediately after the nymphal-adult ecdysis, succinate-cytochrome c reductase increases in activity and continues to rise for about 10 days when it plateaus at the adult level. Cytochrome oxidase activity remains constant during the first 5 days of this period, but, after the fifth day. 

Further studies showed that, in addition to cytochromes a and b, cytochrome c, was lacking, and that the cells contained no NADH or succinate cytochrome c reductase activity, but did contain the primary... 

When air is admitted to an anaerobic yeast culture, respiration reaches normal values within about 4 hours [21]. The cytochrome bands at 556 mfi and 580 m/i are replaced by the bands of cytochromes c, b, and a [19]. Additionally, succinate-cytochrome c reductase activity rises. Catalase and cytochrome peroxidase activities increase over 30-fold, as do NADH-cytochrome c reductase, lactate-cytochrome c reductase, and a-glycerophosphate-cytochrome c reductase. Enzymes of the tricarboxylic acid cycle also increase in activity [21]. 

The half-saturation of the system with cytochrome c was achieved at a concentration of about 10 M (Fig. 15a, b). In all cases, the effect was reversed and prevented by antimycin - an inhibitor of the succinate-cytochrome c reductase activity of this enzyme complex - but was not suppressed by cyanide. In concluding this series of experiments, we investigated the isolated NADH-CoQ reductase complex of the respiratory chain of mitochondria. As can be seen from the results given in Fig. 16, an increase in the concentration of the enzyme complex was accompanied by an increase in the negative charge of the octane phase. 

Figure 3. DeDuve plots of relative specific activities (specific activity/specific activity in PNS) in various fractions. Key to fractions PNS, postnuclear supernatant SOL, soluble MIT, mitochondria MIC, microsomes and Nl, N2, N3, and N4, the discontinuous gradient fractions (see Figure 2). Key to activities A, 5 -nucleotidase B, succinate-cytochrome c reductase and C, NADPH-cytochrome...

Finally, it may be useful to note that the effect of a-tocopherol in restoring the activity of isooctane-extracted DPN- and succinate-cytochrome c reductase systems (Nason and Lehman, 1956) can be duplicated by several members of the ubiquinone series (Weber et al., 1958). 

Similar results have been obtained with mitochondrial fractions from eggs and whole embryos of the surf clam Spisula solidissima. Specific activities of cytochrome oxidase, succinate-cytochrome c reductase, and NADH-cytochrome c reductase were found to be constant during the period from fertilization through the development of swimming stages. Cell division and differentiation apparently entailed neither a significant increase in the total content of. . . mitochondria nor a differentiation of these particles with respect to (their) enzymatic components (Stritt-... 

Fig. 2. Specific activities (Mmoles substrate/minute/mg protein) of cytochrome oxidase and succinate-cytochrome c reductase of mitochondria from embryos of Xenopus laevis. Numerals above abscissa denote Nieuwkoop and Faber stages.

The role of the mitochondrial genome in the synthesis of succinate cytochrome c reductase an inner mitochondrial membrane enzyme, is unclear. The activity of this enzyme decreases in suspension cnltnres of HeLa cells and L cells in the presence of CAP (King et al., 1972). Thus, this enzyme may be added to the list of proteins potentially regulated by the mitochondrial genome. 

DNA is probably the most typical of all reference compounds more than 90% of DNA is in the nuclear pellet, and the small amount in other fractions results from contamination. Eighty per cent of cytochrome oxidase or succinic cytochrome c reductase is associated rith the mitochondrial fraction. By counting the mitochondrria in the nuclear fraction, Schneider and Hogeboom [19] demonstrated that the cytochrome oxidase activity measured in this pellet could be accounted for by contaminating mitochondria. 

NADH cytochrome c reductase was isolated from pigeon breast and pig heart muscle. The enzyme was shown to contain four atoms of iron per flavin molecule. NADH cytochrome c reductase, like succinic dehydrogenase, is a ferroflavoprotein. The ratio of iron to flavin is four. The enzyme contains sulfhydryl groups that can be titrated by classical methods, but their oxidation has no effect on the enzymatic activity. In contrast, the removal of the metal leads to a decrease in the ability of the enzyme to reduce cytochrome c. As for succinic dehydrogenase, the structure of the flavin in NADH cytochrome c reductase is not clear. It was demonstrated that it is not flavin mononucleotide, but the identity of the flavin component with flavin adenine dinucleotide is not established in fact, the flavin component differs from the classical FAD by its chromatographic properties and its behavior in enzymic assays. It is not known if it is a structural variation of the flavin nucleotide or if the nucleotide is conjugated to a peptide. 

The respiratory chain can be separated by various techniques into three multienzyme complexes (Figure 16.3). Complex I is NADH-ubiquinone reductase. Complex III is known as ubiquinone-cytochrome c reductase and contains cytochromes b and Ci. Complex IV is cytochrome oxidase. (Succinate dehydrogenase is referred to as Complex II). Ubiquinone and cytochrome c are small molecules which do not form part of these complexes. Reconstitution of the isolated Complexes I-IV with cytochrome c and ubiquinone leads to recovery of the activity of the respiratory chain. 

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