Resolution and reconstitution of succinate-cytochrome c reductase: preparations and properties of high purity succinate dehydrogenase and ubiquinol-cytochrome c reductase.

An improved method was developed to sequentially fractionate succinate-cytochrome c reductase into three reconstitutive active enzyme systems with good yield: pure succinate dehydrogenase, ubiquinone-binding protein fraction and a highly purified ubiquinol-cytochrome c reductase (cytochrome b-c1 III complex). An extensively dialyzed succinate-cytochrome c reductase was first separated into a succinae dehydrogenase fraction and the cytochrome b-c1 complex by alkali treatment. The resulting succinate dehydrogenase fraction was further purified to homogeneity by the treatment of butanol, calcium phosphate gel adsorption and ammonium sulfate fractionation under anaerobic condition in the presence of succinate and dithiothreitol. The cytochrome b-c1 complex was separated into chtochrome b-c1 III complex and ubiquinone-binding protein fractions by careful ammonium acetate fractionation in the presence of deoxycholate. The purified succinate dehydrogenase contained only two polypeptides with molecular weights of 70 000 anbd 27 000 as revealed by the sodium dodecyl sulfate polyacrylamide gel electrophoretic pattern. The enzyme has the reconstitutive activity and a low Km ferricyanide reductase activity of 85 mumol succinate oxidized per min per mg protein at 38 degrees C. Chemical composition analysis of cytochrome b-c1 III complex showed that the preparation was completely free of contamination of succinate dehydrogenase and ubiquinone-binding protein and was 30% more pure than the available preparation. When these three components were mixed in a proper ratio, a thenoyltrifluoroacetone- and antimycin A-sensitive succinate-cytochrome c reductase was reconstituted.

The prosthetic groups in succinate dehydrogenase. Number and stoichiometry.

I. Succinate:Q oxidoreductase (EC 1.3.99.1) as present in beef-heart submitochondrial particles contains equal amounts of FAD, a [2Fe-2S] cluster and a [4Fe-4S] cluster. Both Fe-S clusters are reducible by succinate. 2. A second type of [2Fe-2S] cluster, called center S-2, that has been proposed to be present in purified preparations of succinate dehydrogenase and isolated Complex II (Ohnishi, T., Winter, D.B., Lim, J. and King, T.E. (1973) Biochem. Biophys. Res. Commun. 53, 231--237) is an artifact introduced by the purification procedure. 3. It is suggested that the 70 000 dalton subunit which is known to bind the flavin, accomodates also the [4Fe-4S] cluster whereas the 28 000 dalton subunit contains the [2Fe-2S] cluster.

The isolation and purification of cytochrome c1 from bovine heart.

A large-scale isolation method for cytochrome c1 from beef heart is presented, based in principle on the procedure of Yu et al. (Yu, C.A., Yu, L. and King, T.E. (1972) J. Biol. Chem. 247, 1012--1019). Optimal solubilization of cytochrome c1 from succinate-cytochrome c oxidoreductase was achieved with 15% beta-mercaptoethanol, 1.5% cholate, 0.5% deoxycholate in 8% saturated ammoniun sulphate. The protein is purfied to a higher degree by chromatography on DEAE-cellulose and Ultrogel AcA 44. The method is reproducible and gives highly purified cytochrome c1 with a yield from succinate-cytochrome c oxidoreductase of 40%. The purified cytochrome c1 contains 32 nmol of heme/mg protein and has a spectral heme-to-protein ratio (Ared417nm/Ax276nm) of 2.7. Reduced cytochrome c1 is oxidized very rapidly by ferricytochrome c (k = 3 . 10(7) M-1 . S-1 at 10 degrees C, 100 mM potassium phosphate (pH 7.0) and 1% Tween 20). Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate shows that the isolated protein consists of one peptide, with a molecular weight of 31 000, carrying the chromophore. In the presence of 1% sodium cholate or 1% Tween 80, cytochrome c1 is in the monomeric state, whereas at lower concentrations of detergent the protein aggregates. The aggregation of cytochrome c1 is found to be reversible.

Succinate-cytochrome c reductase: assessment of its value in the investigation of defects of the respiratory chain.

Defects of the respiratory chain are important causes of human disease and one of the most commonly used assays in the investigation of these patients is the measurement of succinate-cytochrome c reductase. However, this assay measures several components of the respiratory chain and the ability to detect a partial defect in one enzyme complex will depend on the amount of control exerted by that enzyme step on overall electron flux. We show that measurement of succinate-cytochrome c reductase activity may fail to detect partial defects of complex III and therefore is of limited diagnostic value in the identification of complex III defects. However, complex II is a major point of control of flux through succinate-cytochrome reductase and it is likely that measurement of the latter will detect defects of complex II.

Impairment of cellular redox status and membrane protein activities in kidneys from rats with ischemic acute renal failure.

Cellular redox status and membrane protein activities were analyzed in kidneys from rats with ischemic acute renal failure (ARF). ARF was induced by clamping the left renal artery for 50 min. A parallel group of control animals was processed. In the ischemic group urea plasma levels were statistically increased as compared with the control group. Studies employing whole kidney homogenates revealed that ischemia produces an increment in lipid peroxidation levels and a reduction in glutathione concentration and in superoxide dismutase and glutathione peroxidase activities. Since lipid peroxidation may alter the function of membrane proteins we determined succinate cytochrome c reductase (SuccR), sodium-potassium ATPase (Na-K-ATPase), glucose-6-phosphatase (G-6-Pase) and alkaline phosphatase (ALP) activities in whole renal homogenates. Only G-6-Pase and ALP activities were modified by ischemia. Since ALP is a brush border membrane (BBM) enzyme and BBM is one of the main target structures in ARF, we assessed some parameters of BBM functionality. ALP, gamma-glutamyl transferase (gamma-GT) and 5'-nucleotidase (5'-NT) showed diminished activities in BBM from ischemic kidneys. Ischemia also modified the Vmax of paraaminohippuric acid (PAH) uptake without altering Km. An increment of lipid peroxidation and membrane fluidity in BBM was observed after the treatment. Total membrane proteins and protein recoveries in BBM were similar in both experimental groups. Sialic acid and sulfhydryl levels were similar in BBM from ischemic kidney and control ones. In summary, ARF induced by renal artery clamping for 50 min takes place with a significant increase in urea plasma levels. A decrease in the antioxidant defense system is detected. This induces lipid peroxidation in whole renal tissue, which may justify the diminished activities of some membrane enzymes such as G-6-Pase and ALP. A specific analysis of BBM function reveals a significant increment of lipid peroxidation which may be the cause of an increased membrane fluidity. This latter parameter might be, at least in part, responsible for the damaged function of apical ALP, 5'-NT, gamma-GT and PAH carrier.

Muscle coenzyme Q10 concentrations in patients with probable and definite diagnosis of respiratory chain disorders.

Coenzyme Q(10) (CoQ) deficiency syndrome is a disorder of unknown ethiology that may cause different forms of mitochondrial encephalomyopathy. In the present study our aim was to analyse CoQ concentration and mitochondrial respiratory chain (MRC) enzyme activities in muscle biopsies of patients with clinical suspicion and/or biochemical-molecular diagnosis of a mitochondrial disorder. We studied 36 patients classified into 3 groups: 1) 14 patients without a definitive diagnosis of mitochondrial disease, 2) 13 patients with decreased CI + III and II + III activities of the MRC, and 3) 9 patients with definitive diagnosis of mitochondrial disease. Only 1 of the 14 patients of group 1 showed slightly reduced CoQ values in muscle. Six of the 13 patients from group 2 showed partial CoQ deficiency in muscle and 1 of the 9 cases from group 3 presented a slight CoQ deficiency. Significantly positive correlation was observed between CI + III and CII + III activities with CoQ concentrations in the 36 muscle homogenates from patients (r = 0.555; p = 0.001; and r = 0.460; p = 0.005, respectively). In conclusion, measurement of MRC enzyme activities is a useful tool for the detection of CoQ deficiency, which should be confirmed by CoQ quantification.

Partial cytochrome oxidase (aa3) deficiency in chronic progressive external ophthalmoplegia. Histochemical and biochemical studies.

Biochemical and histochemical studies were carried out on 2 patients with chronic progressive external ophthalmoplegia (CPEO). Histological examination revealed prominent ragged-red fibres in the Gomori trichrome stain and cytochrome oxidase staining revealed partial depletion of cytochrome oxidase with negative staining in some fibres with prominent subsarcolemmal mitochondrial aggregations. Polarographic studies with isolated intact skeletal muscle mitochondria revealed low State III respiration rates with NAD- and FAD-linked substrates. Cytochrome aa3 levels were depressed in the one case where a cytochrome difference spectra was recorded. Cytochrome oxidase levels were greatly depressed in muscle homogenate, whereas monoamine oxidase levels were in the normal range, indicating a selective depletion of the former enzyme complex. It is possible that deficiency of cytochrome oxidase may arise as an epiphenomenon in degenerating mitochondria rather than a primary deficiency.

Multiple deletions of mitochondrial DNA in sporadic and atypical cases of encephalomyopathy.

Multiple deletions of mitochondrial DNA (mtDNA) were first identified in patients with mitochondrial encephalomyopathy with a clear mendelian inheritance. We found this genetic alteration in four atypical and sporadic cases of mitochondrial encephalomyopathy, characterized by RRF and partial COX deficiency. One patient was affected by essential hyperCPKemia, 1 by subacute onset flaccid tetraplegia and 2 by parkinsonism. Southern blot and PCR revealed mtDNA multiple deletions in muscle tissue of these patients. These findings indicate that these alterations are not confined to the families with mendelian transmission, but can be present in sporadic cases with heterogeneous phenotypic features.

Mechanisms in 2-hexanone potentiation of chloroform hepatotoxicity.

2-Hexanone (2-Hx) is known to potentiate chloroform (CHCl3) hepatotoxicity in part by increasing the bioactivation of CHCl3 to phosgene (COCl2). Treatment of rats with 2-Hx + CHCl3 in vivo did not initiate peroxidation of hepatic fatty acids as determined by formation of conjugated dienes or depletion of unsaturated fatty acids, or as determined by production of malondialdehyde (MDA) in vitro. A 5-fold decrease in the specific activity of succinate-dependent cytochrome c reductase in liver from rats treated in vivo with corn oil (vehicle) + CHCl3 and in rats treated with 2-Hx + CHCl3 indicated that a mechanism independent of CHCl3 bioactivation may add to the hepatotoxic effects which result from the metabolism of chloroform to phosgene.

Metabolic differentiation of bloodstream forms of Trypanosoma brucei brucei into procyclic forms. Effect of hydroxyurea, arabinosyl adenine, and serum omission.

Bloodforms of Trypanosoma brucei brucei STIB 247 taken from rats and containing more than 80 per cent short stumpy forms, differentiated in vitro to procyclic forms in medium SDM 79 (Brun and Schönenberger 1979), enriched with 3 mmol.dm-3 cis-aconitate. Cell division was abolished by the addition of hydroxyurea (200 micrograms.ml-1) or arabinosyl adenine (20 micrograms.ml-1 to the cultivation medium, or by the omission of serum from the medium. The ultrastructure of exponentially growing controls was rearranged within 24 h. The endogenous respiration and the respiration stimulated by proline, succinate, and 2-oxoglutarate were detectable within 12 h; after 48 h the respiration rates were comparable to those found in the established procyclic forms. After 12 h the respiration was inhibited by 200 mumol.dm-3 KCN, and by 20 mumol.dm-3 antimycin to the extent found in procyclic forms. Hydroxyurea did not significantly affect respiration. Activities of procyclic-stage enzyme markers malate dehydrogenase, threonine dehydrogenase, succinate: cytochrome c reductase, and NADH: cytochrome c reductase rose within 48 h of differentiation to values which were close to those found in established procyclic forms. The activity of glutamate dehydrogenase (NAD-specific), however, was only 1/3 of that in the procyclics, and no citrate synthase was detected in differentiating culture. Glycosomal malate dehydrogenase was detected after 6 h. In the presence of hydroxyurea or arabinosyl adenine, or in the absence of serum, respiration rates, marker enzyme activities, and glycosomal malate dehydrogenase developed to the extent comparable to the untreated controls. The results suggest that it is possible to separate the process of differentiation from cell proliferation. Cell division is not a necessary prerequisite of differentiation.

[Characterization of mitochondrial myopathies through the evaluation of the enzymatic activities involved in energy metabolism]. / Caracterização de miopatias mitocondriais através da avaliação das atividades enzimáticas envolvidas no metabolismo energético.

The activities of the enzymes NADH dehydrogenase, NADH cytochrome e reductase, succinate dehydrogenase, succinate cytochrome e reductase, cytochrome c oxidase and citrate synthase in normal and sick human skeletal muscle mitochondria were determined. A control group was formed by 13 normal people and without using continuous medication. The patient group was formed by 10 people whose pathological diagnosis indicated suspicion of mitochondrial myopathy. A decrease in the activity of the enzymes in all patient was observed: 7 with abnormality in all the tested enzymes; 2 with deficiencies in all the enzymes except cytochrome e oxidase; and 1 with dysfunction only in the activities of succinate dehydrogenase and succinate cytochrome e reductase. The results indicate multiple or combined deficiencies in the respiratory chain, besides dysfunction of citrate synthase in 9 patients. In one exceptional case, the enzymatic deficiency was restricted to complex II. It is possible to conclude that the methodology used herein is adequate and easily applicable to clinical objectives, and that the results obtained allow characterization of the deficient mitochondrial enzymatic complexes, thus showing that the origin of the diseases is an energetic metabolic dysfunction.

[Fractionation of inner and outer mitochondrial membranes in rat liver (author's transl)].

Mitochondrial membranes of rat liver were fractionated into inner and outer membranes by osmotic shock. Purity of the inner and outer membrane subfractions was examined by estimating marker enzyme activities of succinate-cytochrome c reductase and monoamine oxidase, respectively. Monoamine oxidase activity in the outer membrane subfraction was found to be 21.90 mmumoles of benzaldehyde produced/min./mg. protein and 1.57 mmumoles in the inner membranes, whereas succinate-cytochrome c reductase activity was 13.4 and 33.0 mmumoles/min./mg. protein, respectively. These results indicate higher purity of each membrane fraction obtained.

Human skeletal muscle mitochondria in aging: lack of detectable morphological and enzymic defects.

We have investigated structural and functional properties of skeletal muscle mitochondria obtained from biopsies from young and old individuals. The morphometric analysis of muscle sections revealed a tendency to an increase of total area, numerical density and volume density of mitochondria in the aged. The enzymatic activities of NADH-Coenzyme Q reductase, succinate cytochrome c reductase, ubiquinol-cytochrome c reductase exhibited a high variability of specific activities without any correlation with age. Expression of the values as enzyme turnovers reduced the variability but was unable to reveal any age-dependent modification.

A mitochondrial myopathy with a defective respiratory chain and carnitine deficiency.

A boy presented suffering from generalised weakness, exercise intolerance and lactic acidosis. The weakness became evident at 2 years. A cerebral CT-scan showed cerebellar atrophy and central and peripheral atrophy of both hemispheres. With trichrome staining about 20% of the muscle fibres showed large areas containing red-staining granular material. Electron microscopic examination showed that this material consisted of areas of mitochondrial proliferation, most of the mitochondria having abnormal ultrastructural characteristics. Pyruvate dehydrogenase complex and citric acid cycle activities were determined by measuring 14CO2 production from various labelled substrates. Diminished oxidation rates were found with the patient's muscle homogenate for all substrates tested, indicating a defect in the respiratory chain. The cytochromes were present in normal quantities. Succinate cytochrome c reductase activity was very decreased. Carnitine concentration was decreased in serum and in muscle as well.

Respiratory chain enzyme defects in patients with idiopathic inflammatory myopathy.

OBJECTIVE: To analyse muscle respiratory chain enzymes in idiopathic inflammatory myopathy. METHODS: Four consecutive female patients seen at our hospital with idiopathic inflammatory myopathy were studied. Muscle histochemical staining included NADH tetrazolium reductase and succinate dehydrogenase tests. Activity of rotenone sensitive NADH cytochrome c reductase (complex I and III) succinate dehydrogenase (complex II), succinate cytochrome c reductase (complex II and III), cytochrome c oxidase (complex IV), and citrate synthase (a mitochondrial matrix enzyme), was measured spectrophotometrically in muscle homogenates. Free carnitine, and short and long chain acylcarnitine esters were determined in muscle homogenates by a radiochemical procedure. RESULTS: Three patients had mitochondrial proliferation in nonregenerating muscle fibres; these patients had defects of respiratory chain enzyme complexes. Carnitine concentrations, measured in two of the four patients, revealed carnitine deficiency in one. CONCLUSION: Our results suggest that mitochondrial dysfunction may be present in patients with inflammatory myositis.

Use of a fluorescent probe to determine the viscosity of LM cell membranes with altered phospholipid compositions.

The phospholipid compostition of LM cells grown in tissue culture was altered by substituting ethanolamine for choline in the growth medium. The plasma membrane isolated from cells grown in medium conatining ethanolamine for 83 h had a sixfold increase in the ratio of phosphatidylethanolamine to phosphatidylcholine, the two major phospholipid classes. This was accompanied by small changes in other lipid components of the membrane. There was also a sixfold increase in the amount of triacylglycerols and alkyldiacylglycerols which were not associated with the membrane fraction of the cell. No significant changes occurred in the lipid composition of cells during growth in choline containing medium. The viscosity of plasma membranes was studied in whole cells and isolated membranes using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene. Plasma membranes isolated from ethanolamine-supplemented cells had greater viscosities than membranes isolated from choline-supplemented cells. When whole cells were labeled with the fluorescent probe, the opposite trend in the apparent membrane viscosity was observed. This was due primarily to the probe penetrating into nonmembranous neutral lipids rather than remaining localized in the surface membrane of the cells. Since the enthanolamine-supplemented cells contained more low viscosity neutral lipids, the whole cells gave an apparently lower viscosity as compared with choline-supplemented cells, thus, measurements carried out on whole cells gave an inaccurate determination of the viscosity of the surface membrane.

Role of ubiquinone in the mitochondrial generation of hydrogen peroxide.

Antimycin-inhibited bovine heart submitochondrial particles generate O2- and H2O2 with succinate as electron donor. H2O2 generation involves the action of the mitochondrial superoxide dismutase, in accordance with the McCord & Fridovich [(1969) j. biol. Chem. 244, 6049-6055] reaction mechanism. Removal of ubiquinone by acetone treatment decreases the ability of mitochondrial preparations to generate O2- and H2O2, whereas supplementation of the depleted membranes with ubiquinone enhances the peroxide-generating activity in the reconstituted membranes. Addition of superoxide dismutase to ubiquinone-reconstituted membranes is essential in order to obtain maximal rates of H2O2 generation since the acetone treatment of the membranes apparently inactivates (or removes) the mitochondrial superoxide dismutase. Parallel measurements of H2O2 production, succinate dehydrogenase and succinate-cytochrome c reductase activities show that peroxide generation by ubiquinone-supplemented membranes is a monotonous function of the reducible ubiquinone content, whereas the other two measured activities reach saturation at relatively low concentrations of reducible quinone. Alkaline treatment of submitochondrial particles causes a significant decrease in succinate dehydrogenase activity and succinate-dependent H2O2 production, which contrasts with the increase of peroxide production by the same particles with NADH as electron donor. Solubilized succinate dehydrogenase generates H2O2 at a much lower rate than the parent submitochondrial particles. It is postulated that ubisemiquinone (and ubiquinol) are chiefly responsible for the succinate-dependent peroxide production by the mitochondrial inner membrane.

MTO1 codes for a mitochondrial protein required for respiration in paromomycin-resistant mutants of Saccharomyces cerevisiae.

Mutations in MTO1 express a respiratory defect only in the context of a mitochondrial genome with a paromomycin-resistance allele. This phenotype is similar to that described previously for mss1 mutants by Decoster, E., Vassal, A., and Faye, G. (1993) J. Mol. Biol. 232, 79-88. We present evidence that Mto1p and Mss1p are mitochondrial proteins and that they form a heterodimer complex. In a paromomycin-resistant background, mss1 and mto1 mutants are inefficient in processing the mitochondrial COX1 transcript for subunit 1 of cytochrome oxidase. The mutants also fail to synthesize subunit 1 and show a pleiotropic absence of cytochromes a, a3, and b. In vivo pulse labeling of an mto1 mutant, however, indicate increased rates of synthesis of other mitochondrial translation products. The respiratory defective phenotype of mto1 and mss1 mutants is not seen in a paromomycin-sensitive genetic background. The visible absorption spectra of such strains indicate a higher ratio of cytochromes b/a and elevated NADH- and succinate-cytochrome c reductase activities. To explain these phenotypic characteristics, we proposed that the Mto1p.Mss1p complex plays a role in optimizing mitochondrial protein synthesis in yeast, possibly by a proofreading mechanism.