Influence of increased environmental temperature on oxidation processes in rat liver mitochondria

Exposure of rats to elevated temperature of 28 degrees C or 35 degrees C for 3 days six hours daily resulted in a decreased rate of oxidation with succinate or glutamate + malate as substrates, by the mitochondria of liver. The higher decrease was observed in environment temperature of 35 degrees C. There was no change in ADP/O ratio. The activities of NADH: cytochrome c reductase and cytochrome oxidase were stimulated but activities of succinate dehydrogenase and succinate cytochrome reductase were decreased.     

Direct interaction between the internal NADH: ubiquinone oxidoreductase and ubiquinol:cytochrome c oxidoreductase in the reduction of exogenous quinones by yeast mitochondria.

The reduction of duroquinone (DQ) and 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (DB) by NADH and ethanol was investigated in intact yeast mitochondria with good respiratory control ratios. In these mitochondria, exogenous NADH is oxidized by the NADH dehydrogenase localized on the outer surface of the inner membrane, whereas the NADH produced by ethanol oxidation in the mitochondrial matrix is oxidized by the NADH dehydrogenase localized on the inner surface of the inner membrane. The reduction of DQ by ethanol was inhibited 86% by myxothiazol; however, the reduction of DQ by NADH was inhibited 18% by myxothiazol, suggesting that protein-protein interactions between the internal (but not the external) NADH: ubiquinone oxidoreductase and ubiquinol:cytochrome c oxidoreductase (the cytochrome bc1 complex) are involved in the reduction of DQ by NADH. The reduction of DQ and DB by NADH and ethanol was also investigated in mutants of yeast lacking cytochrome b, the iron-sulfur protein, and ubiquinone. The reduction of both quinone analogues by exogenous NADH was reduced to levels that were 10 to 20% of those observed in wild-type mitochondria; however, the rate of their reduction by ethanol in the mutants was equal to or greater than that observed in the wild-type mitochondria. Furthermore, the reduction of DQ in the cytochrome b and iron-sulfur protein lacking mitochondria was myxothiazol sensitive, suggesting that neither of these proteins is an essential binding site for myxothiazol. The mitochondria from the three mutants also contained significant amounts of antimycin- and myxothiazol-insensitive NADH:cytochrome c reductase activity, but had no detectable succinate:cytochrome c reductase activity. These results suggest that the mutants lacking a functional cytochrome bc1 complex have adapted to oxidize NADH.     

Mutations affecting the reduced nicotinamide adenine dinucleotide dehydrogenase complex of Escherichia coli

A strain carrying a point mutation affecting the NADH dehydrogenase complex of Escherichia coli has been isolated and its properties examined. The gene carrying the mutation (designated ndh) was located on the E. coli chromosome at about minute 23 and was shown to be cotransducible with the pyrC gene. Strains carrying the ndh^? allele were found to be unable to grow on mannitol and to grow very poorly on glucose unless the medium was supplemented with succinate, acetate or casamino acids.The following properties of strains carrying the ndh^? allele were established which suggest that the mutation affects the NADH dehydrogenase complex but apparently not the primary dehydrogenase. Membrane preparations possess normal to elevated levels of d-lactate oxidase and succinate oxidase activities but NADH oxidase is absent. NADH is unable to reduce ubiquinone in the aerobic steady state and reduces cytochrome b very slowly when the membranes become anaerobic. NADH dehydrogenase, measured as NADH-dichlorophenolindophenol reductase is reduced but not absent. NADH oxidase is stimulated by menadione although not by Q-3 or MK-1 and in the presence of menadione, cytochrome b is reduced normally by NADH.Further mutants affected in NADH oxidase were isolated using a screening procedure based on the growth characteristics of the original ndh^? strain. The mutations carried by these strains were all cotransducible with the pyrC gene and the biochemical properties of the additional mutants were similar to those of the original mutant.The properties of the group of ndh^? mutants established so far suggest that they are affected in the transfer of reducing equivalents from the NADH dehydrogenase complex to ubiquinone.     

Isolation and characterization of complex I, rotenone-sensitive NADH: ubiquinone oxidoreductase, from the procyclic forms of Trypanosoma brucei.

Additional characterization of complex I, rotenone-sensitive NADH:ubiquinone oxidoreductase, in the mitochondria of Trypanosoma brucei brucei has been obtained. Both proline:cytochrome c reductase and NADH:ubiquinone oxidoreductase of procyclic T. brucei were inhibited by the specific inhibitors of complex I rotenone, piericidin A, and capsaicin. These inhibitors had no effect on succinate: cytochrome c reductase activity. Antimycin A, a specific inhibitor of the cytochrome bc1 complex (ubiquinol:cytochrome c oxidoreductase), blocked almost completely cytochrome c reductase activity with either proline or succinate as electron donor, but had no inhibitory effect on NADH:ubiquinone oxidoreductase activity. The rotenone-sensitive NADH:ubiquinone oxidoreductase of procyclic T. brucei was partially purified by sucrose density centrifugation of mitochondria solubilized with dodecyl-beta-D-maltoside, with an approximately eightfold increase in specific activity compared to that of the mitochondrial membranes. Four polypeptides of the partially purified enzyme were identified as the homologous subunits of complex I (51 kDa, PSST, TYKY, and ND4) by immunoblotting with antibodies raised against subunits of Paracoccus denitrificans and against synthetic peptides predicted from putative complex I subunit genes encoded by mitochondrial and nuclear T. brucei DNA. Blue Native polyacrylamide gel electrophoresis of T. brucei mitochondrial membrane proteins followed by immunoblotting revealed the presence of a putative complex I with a molecular mass of 600 kDa, which contains a minimum of 11 polypeptides determined by second-dimensional Tricine-SDS/PAGE including the 51 kDa, PSST and TYKY subunits.     

Glucagon treatment of rats activates the respiratory chain of liver mitochondria at more than one site

The rate of reduction of ferricyanide in the presence and absence of antimycin and ubiquinone-1 was measured using liver mitochondria from control and glucagon treated rats. Glucagon treatment was shown to increase electron flow from both NADH and succinate to ubiquinone, and from ubiquinone to cytochrome c. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was shown to inhibit the oxidation of glutamate + malate to a much greater extent than that of succinate or duroquinol. Spectral and kinetic studies confirmed that electron flow between NADH and ubiquinone was the primary site of action but that the interaction of the ubiquinone pool with complex 3 was also affected. The effects of various respiratory chain inhibitors on the rate of uncoupled oxidation of succinate and glutamate + malate by control and glucagon treated mitochondria were studied. The stimulation of respiration seen in the mitochondria from glucagon treated rats was maintained or increased as respiration was progressively inhibited with DCMU, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), 2-heptyl-4-hydroxyquinoline-n-oxide (HQNO) and colletotrichin, but greatly reduced when inhibition was produced with malonate or antimycin. These data were also shown to support the conclusion that glucagon treatment may cause some stimulation of electron flow through NADH dehydrogenase, succinate dehydrogenase and through the bc1 complex, probably at the point of interaction of the complexes with the ubiquinone pool. The effects of glucagon treatment on duroquinol oxidation and the inhibitor titrations could not be mimicked by increasing the matrix volume, nor totally reversed by aging of mitochondria. These are both processes that have been suggested as the means by which glucagon exerts its effects on the respiratory chain (Armston, A.E., Halestrap, A.P. and Scott, R.D., 1982, Biochim. Biophys. Acta 681, 429-439). It is concluded that an additional mechanism for regulating electron flow must exist and a change in lipid peroxidation of the inner mitochondrial membrane is suggested.     

Changes in Terminal Respiratory Pathways of Bacillus subtilis During Germination, Outgrowth, and Vegetative Growth

The chemical and enzymatic properties of the cytochrome system in the particulate preparations obtained from dormant spores, germinated spores, young vegetative cells, and vegetative cells of Bacillus subtilis PCI219 were investigated. Difference spectra of particulate fractions from dormant spores of this strain suggested the presence of cytochromes a, a(3), b, c(+c(1)), and o. All of the cytochrome components were present in dormant spores and in germinated spores and vegetative cells at all stages which were investigated. Concentrations of cytochromes a, a(3), b, and c(+c(1)) increased during germination, outgrowth, and vegetative growth, but that of cytochrome o was highest in dormant spores. As the cytochrome components were reducible by reduced nicotinamide adenine dinucleotide (NADH), they were believed to be metabolically active. Difference spectra of whole-cell suspensions of dormant spores and vegetative cells were coincident with those of the particulate fractions. NADH oxidase and cytochrome c oxidase were present in dormant spores, germinated spores, and vegetative cells at all stages after germination, but succinate cytochrome c reductase was not present in dormant spores. Cytochrome c oxidase and succinate cytochrome c reductase activities increased with growth, but NADH oxidase activity was highest in germinated spores and lowest in vegetative cells. There was no striking difference between the effects of respiratory inhibitors on NADH oxidase in dormant spores and those on NADH oxidase in vegetative cells.     

Membrane-bound respiratory of Spirillum itersonii.

The membrane-bound respiratory system of the gram-negative bacterium Spirillum itersonii was investigated. It contains cytochromes b (558), c (550), and o (558) and beta-dihydro-nicotinamide adenine dinucleotide (NADH) and succinate oxidase activities under all growth conditions. It is also capable of producing D-lactate and alpha-glycerophosphate dehydrogenases when grown with lactate or glycerol as sole carbon source. Membrane-bound malate dehydrogenase was not detectable under any conditions, although there is high activity of soluble nicotinamide adenine dinucleotide: malate dehydrogenase. When grown with oxygen as the sole terminal electron acceptor, approximately 60% of the total b-type cytochrome is present as cytochrome o, whereas only 40% is present as cytochrome o in cells grown with nitrate in the presence of oxygen. Both NADH and succinate oxidase are inhibited by azide, cyanide, antimycin A, and 2-n-heptyl-4-hydroxyquinoline-N-oxidase at low concentrations. The ability of these inhibitors to completely inhibit oxidase activity at low concentrations and their effects upon the aerobic steady-state reduction levels of b- and c-type cytochromes as well as the aerobic steady-state reduction levels obtained with NADH, succinate, and ascorbate-dichlorophenolindophenol suggest that presence of an unbranched respiratory chain in S. itersonii with the order ubiquinone leads to b leads to c leads to c leads to oxygen.     

The oxidative inactivation of mitochondrial electron transport chain components and ATPase

Bovine heart submitochondrial particles (SMP) were exposed to continuous fluxes of hydroxyl radical (.OH) alone, superoxide anion radical (O2-) alone, or mixtures of .OH and O2-, by gamma radiolysis in the presence of 100% N2O (.OH exposure), 100% O2 + formate (O2- exposure), or 100% O2 alone (.OH + O2- exposure). Hydrogen peroxide effects were studied by addition of pure H2O2. NADH dehydrogenase, NADH oxidase, succinate dehydrogenase, succinate oxidase, and ATPase activities (Vmax) were rapidly inactivated by .OH (10% inactivation at 15-40 nmol of .OH/mg of SMP protein, 50-90% inactivation at 600 nmol of .OH/mg of SMP protein) and by .OH + O2- (10% inactivation at 20-80 nmol of .OH + O2-/mg of SMP protein, 45-75% inactivation at 600 nmol of .OH + O2-/mg of SMP protein). Importantly, O2- was a highly efficient inactivator of NADH dehydrogenase, NADH oxidase, and ATPase (10% inactivation at 20-50 nmol of O2-/mg of SMP protein, 40% inactivation at 600 nmol of O2-/mg of SMP protein), a mildly efficient inactivator of succinate dehydrogenase (10% inactivation at 150 nmol of O2-/mg of SMP protein, 30% inactivation at 600 nmol of O2-/mg of SMP protein), and a poor inactivator of succinate oxidase (less than 10% inactivation at 600 nmol of O2-/mg of SMP protein). H2O2 partially inactivated NADH dehydrogenase, NADH oxidase, and cytochrome oxidase, but even 10% loss of these activities required at least 500-600 nmol of H2O2/mg of SMP protein. Cytochrome oxidase activity (oxygen consumption supported by ascorbate + N,N,N',N'-tetramethyl-p-phenylenediamine) was remarkably resistant to oxidative inactivation, with less than 20% loss of activity evident even at .OH, O2-, OH + O2-, or H2O2 concentrations of 600 nmol/mg of SMP protein. Cytochrome c oxidase activity, however (oxidation of, added, ferrocytochrome c), exhibited more than a 40% inactivation at 600 nmol of .OH/mg of SMP protein. The .OH-dependent inactivations reported above were largely inhibitable by the .OH scavenger mannitol. In contrast, the O2(-)-dependent inactivations were inhibited by active superoxide dismutase, but not by denatured superoxide dismutase or catalase. Membrane lipid peroxidation was evident with .OH exposure but could be prevented by various lipid-soluble antioxidants which did not protect enzymatic activities at all.(ABSTRACT TRUNCATED AT 400 WORDS)     

Electron-transfer complexes of Ascaris suum muscle mitochondria. II. Succinate-coenzyme Q reductase (complex II) associated with substrate-reducible cytochrome b-558

A succinate-coenzyme Q reductase (complex II) was isolated in highly purified form from Ascaris muscle mitochondria by detergent solubilization, ammonium sulfate fractionation and gel filtration on a Sephadex G-200 column. The enzyme preparation catalyzes electron transfer from succinate to coenzyme Q1 with a specific activity of 1.2 mumol coenzyme Q1 reduced per min per mg protein at 25 degrees C. The isolated complex II is essentially free of NADH-ferricyanide reductase, reduced CoQ2-cytochrome c reductase and cytochrome c oxidase and consists of four major polypeptides with apparent molecular weights of 66 000, 27 000, 12 000 and 11 000 and two minor ones with Mr of 36 000 and 16 000. The complex II contained cytochrome b-558, a major constituent cytochrome of Ascaris mitochondria, at a concentration of 3.6 nmol per mg protein, but neither other cytochromes nor quinone. The cytochrome b-558 in the complex II was reduced with succinate. In the presence of Ascaris NADH-cytochrome c reductase (complex I-III) (Takamiya, S., Furushima, R. and Oya, H. (1984) Mol. Biochem. Parasitol. 13, 121-134), the cytochrome b-558 in complex II was also reduced with NADH and reoxidized with fumarate. These results suggest the cytochrome b-558 to function as an electron carrier between NADH dehydrogenase and succinate dehydrogenase in the Ascaris NADH-fumarate reductase system.     

Oxidative activities in mitochondria-like particles from Setaria digitata, a filarial parasite.

The oxidative metabolic potential of Setaria digitata, a filarial parasite found in the intraperitoneal cavity of cattle, was investigated. These worms showed active wriggling movements which were not affected by respiratory poisons such as cyanide, rotenone and malonate. They also possessed cyanide-insensitive and glucose-independent oxygen consumption pathways. By differential centrifugation of sucrose homogenates, a fraction containing mitochondria-like particles was obtained in which the activity of the marker enzyme, succinate dehydrogenase, was recovered. This fraction catalysed succinate- and NADH-dependent reduction of both cytochrome c and dyes. Oxygen uptake found with succinate, NADH and ascorbate as substrates was not sensitive to cyanide. Cytochromes could not be detected in either this fraction or homogenates of the worms. H2O2 generation with a number of substrates and lipid peroxidation by measuring malondialdehyde formed as well as by accompanying oxygen uptake were demonstrated in the mitochondria-like particles. A lipid quinone, possibly with a short side chain and related to ubiquinone, was detected in the worms. The results suggested the existence of two cyanide-insensitive oxygen-consuming reactions in Setaria: one respiratory substrate-independent lipid peroxidation, and a second substrate-dependent reaction that requires an auto-oxidizable quinone but not a cytochrome system.     

Effect of succinate on mitochondrial lipid peroxidation. 2. The protective effect of succinate against functional and structural changes induced by lipid peroxidation

The damaging effects of ADP/Fe/NADPH-induced lipid peroxidation were studied on the enzymes and membranes of rat liver mitochondria. Succinate, an inhibitor of mitochondrial lipid peroxidation, prevented or delayed most of the damage caused by the peroxidation on different mitochondrial structures and functions. There were marked abnormalities on the electrophoretic pattern of mitochondrial proteins during the course of lipid peroxidation. The disappearance of particular polypeptide bands and the accumulation of high-molecular-weight aggregates could be observed. Succinate was found to delay these effects. As a consequence of lipid peroxidation the succinate oxidase activity of mitochondria was decreased. The succinate dehydrogenase enzyme and the component(s) of the respiratory chain were inactivated. Succinate prevented the inactivation of succinate dehydrogenase but did not protect the other components of terminal oxidation chain. From the matrix enzymes the glutamate dehydrogenase retained its full activity but the NADP-linked isocitrate dehydrogenase was inactivated. The mitochondrial membranes became permeable to large protein molecules. Succinate prevented the inactivation of isocitrate dehydrogenase and delayed the release of protein molecules from mitochondria.     

Effect of the nonionic detergent Triton X-100 on mitochondrial succinate-oxidizing enzymes

Specific activities of succinate:coenzyme Q reductase, ubiquinone:cytochrome c reductase, cytochrome oxidase, succinate:cytochrome c reductase, succinate oxidase, and ubiquinol oxidase have been measured in rat liver mitochondria in the presence of Triton X-100. The last three activities are much more sensitive to Triton X-100 than the first ones; the data suggest that the electron transport chain components cannot react with each other in the presence of the detergent. At least in the case of succinate:cytochrome c reductase, reconstitution of the detergent-treated membranes with externally added phospholipids reverses the inhibition produced by Triton X-100. These results support the idea that the respiratory chain components diffuse at random in the plane of the inner mitochondrial membrane; the main effect of the detergent would be to impair lateral diffusion by decreasing the area of lipid bilayer. When detergent-treated mitochondrial suspensions are centrifuged in order to separate the solubilized from the particulate material, only the first three enzyme activities mentioned above are found in the supernatants. After centrifugation, a latent ubiquinol:cytochrome c oxidase activity becomes apparent, whereas the same centrifugation process produces inhibition of cytochrome c oxidase in the presence of certain Triton X-100 concentrations. These effects could be due either to a selective solubilization of regulatory or catalytic subunits or to a conformational change of the enzyme-detergent complex.     

Influence of oxygen tension on the respiratory activity of Mycobacterium phlei

Growth of Mycobacterium phlei under low oxygen tension resulted in specific activities two to twenty times lower for formate dehydrogenase, malate dehydrogenase, beta-hydroxybutyrate dehydrogenase, lactate oxidase and NADH dehydrogenase than when cultures were grown under high aeration. An increase in fumarate reductase and succinate dehydrogenase occurred with M. phlei grown under low oxygen tension. Malate: vitamin K dehydrogenase and glucose-6-phosphate dehydrogenase activity were not significantly affected by the oxygen tension used to grow the bacteria, and neither culture contained a lactate dehydrogenase. With growth of M. phlei in conditions of low oxygen tension, cytochrome a was not detected, but cytochrome b was prominent in membranes and cytochrome c was present in the soluble fraction.     

Studies on the succinate dehydrogenating system. Isolation and properties of the mitochondrial succinate-ubiquinone reductase

A simple procedure for preparation of highly purified soluble succinate-ubiquinone reductase from bovine heart mitochondrial particles is described. The enzyme exhibits four major bands on sodium dodecyl sulfate gel electrophoresis and contains (nmol per mg protein): covalently bound flavin, 6; non-heme iron, 53; acid-labile sulfur, 50; cytochrome b-560 heme, 1.2. The enzyme catalyzes thenoyltrifluoroacetone, or carboxin-sensitive (pure non-competitive with Q2) reduction of Q2 by succinate with a turnover number close to that in parent submitochondrial particles. The succinate reduced enzyme exhibits ferredoxin-type iron-sulfur center EPR-signal (g = 1.94 species) and a semiquinone signal (g = 2.00). An oxidized preparation shows a symmetric signal centered around g = 2.01. An unusual dissociation of the enzyme in the absence of a detergent is described. When added to the assay mixture from a concentrated protein-detergent solution, the enzyme does not reduce Q2 being highly reactive towards ferricyanide ('low Km ferricyanide reactive site'; Vinogradov, A.D., Gavrikova, E.V. and Goloveshkina, V.G. (1975) Biochem. Biophys. Res. Commun. 65, 1264-1269). The ubiquinone reductase, not the ferricyanide reductase was observed when the enzyme was added to the assay mixture from the diluted protein-detergent solutions. Thus the dissociation of succinate dehydrogenase from the complex occurs in the absence of a detergent dependent on the concentration of the protein-detergent complex in the stock preparation where the samples for the assay are taken from. An active antimycin-sensitive succinate-cytochrome c reductase was reconstituted by admixing of the soluble succinate-ubiquinone reductase and the cytochrome b-c1 complex, i.e., from the complexes which both contain the ubiquinone reactivity conferring protein (QPs). Cytochrome c reductase was also reconstituted from the succinate-ubiquinone reductase and succinate-cytochrome c reductase containing inactivated succinate dehydrogenase. The reconstitution experiments suggest that there exists a specific protein-protein (or lipid) interaction between QPs and a certain component(s) of the b-c1 complex.     

Substrate kinetics of the Acanthamoeba castellanii alternative oxidase and the effects of GMP

In Acanthamoeba castellanii mitochondria, the apparent affinity values of alternative oxidase for oxygen were much lower than those for cytochrome c oxidase. For unstimulated alternative oxidase, the K(Mox) values were around 4-5 microM both in mitochondria oxidizing 1 mM external NADH or 10 mM succinate. For alternative oxidase fully stimulated by 1 mM GMP, the KK(Mox) values were markedly different when compared to those in the absence of GMP and they varied when different respiratory substrates were oxidized (K(Mox) was around 1.2 microM for succinate and around 11 microM for NADH). Thus, with succinate as a reducing substrate, the activation of alternative oxidase (with GMP) resulted in the oxidation of the ubiquinone pool, and a corresponding decrease in K(Mox). However, when external NADH was oxidized, the ubiquinone pool was further reduced (albeit slightly) with alternative oxidase activation, and the K(Mox) increased dramatically. Thus, the apparent affinity of alternative oxidase for oxygen decreased when the ubiquinone reduction level increased either by changing the activator or the respiratory substrate availability.     

Lipid peroxidation and the reduction of ADP-Fe3+ chelate by NADH-ubiquinone reductase preparation from bovine heart mitochondria.

The NADH-ubiquinone reductase preparation (Complex I) of bovine hart mitochondria catalysed in the presence of reduced coenzymes and ADP-Fe3+ the lipid peroxidation of liposomes prepared from mitochondrial lipids. The apparent Km values for the coenzymes and the optimal pH of the reactions agreed well with those of the lipid peroxidation of the submitochondrial particles treated with rotenone. On assay of the reduction of ADP-Fe3+ chelate by the reduction of cytochrome c in the presence of superoxide dismutase and antimycin A or by the oxidation of reduced coenzymes, the reactions were not affected by rotenone but were inhibited by thiol-group inhibitors. The properties of the ADP-Fe3+ reductase activity were highly consistent with those of the lipid-peroxidation reaction. These observations suggest that electrons from reduced coenzymes are transferred to ADP-Fe3+ chelate from a component between a mercurial-sensitive site and the rotenone-sensitive one of the NADH dehydrogenase and that the reduction of ADP-Fe3+ chelate by the NADH dehydrogenase is an essential step in the lipid peroxidation.     

Sequential damage in mitochondrial complexes by peroxidative stress

The biochemical characteristics of the electron transfer chain are evaluated in purified non-synaptic (free) mitochondria from the forebrain of 60-week-old rats weekly subjected to peroxidative stress (once, twice, or three times) by the electrophilic prooxidant 2-cyclohexene-1-one. The following parameters are evaluated: (a) content of respiratory components, namely ubiquinone, cytochrome b, cytochrome c₁, cytochrome c; (b) specific activity of enzymes, namely citrate synthase, succinate dehydrogenase, rotenone-sensitive NADH: cytochrome c reductase, cytochrome oxidase; (c) concentration of reduced glutathione (GSH). Before the first peroxidative stress induction, the rats are administered for 8 weeks by intraperitoneal injection of vehicle, papaverine, -yohimbine, almitrine or hopanthenate. The rats are treated also during the week(s) before the second or third peroxidative stress. The cerebral peroxidative stress induces: (a) initially, a decrease in brain GSH concentration concomitant with a decrease in the mitochondrial activity of cytochrome oxidase of aa₃-type (complex IV), without changes in ubiquinone and cytochrome b populations; (b) subsequently, an alteration in the transfer molecule cytochrome c and, finally, in rotenone-sensitive NADH-cytochrome c reductase (complex I) and succinate dehydrogenase (complex II). The selective sensitivity of the chain components to peroxidative stress is supported by the effects of the concomitant subchronic treatment with agents acting at different biochemical steps. In fact, almitrine sets limits to its effects at cytochrome c content and aa₃-type cytochrome oxidase activity, while -yohimbine sets limits to its effects at the level of tricarboxylic acid cycle (citrate synthase) and/or of intermediary between tricarboxylic acid cycle and complex II (succinate dehydrogenase). The effects induced by sequential peroxidative stress and drug treatment are supportive of the hypothesis that leakage of electrons (as a mandatory side-effect of the normal flux of electrons from both NADH and succinate to molecular oxygen) would be due to alteration in both availability of GSH and the content of components in the respiratory chain associated to energy-transducing system. In this field there is a cascade of derangements involving, at the beginning, the complex IV and, subsequently, other chain components, including cytochrome c and, finally, complexes II and I.     

Pathways of glucose catabolism in procyclic Trypanosoma congolense

Studies of respiration on glucose in procyclic Trypanosoma congolense in the presence of rotenone, antimycin, cyanide, salicylhydroxamic acid and malonate have indicated the presence of NADH dehydrogenase, cytochrome b-c1, cytochrome aa3, trypanosome alternate oxidase and NADH fumarate reductase/succinate dehydrogenase pathway that contributes electrons to coenzyme Q of the respiratory chain. The rotenone sensitive NADH dehydrogenase, the trypanosome alternate oxidase, and cytochrome aa3 accounted for 24.5 +/- 6.5, 36.2 +/- 4.2 and 54.1 +/- 5.5% respectively of the total respiration. Activities of lactate dehydrogenase, NAD(+)-linked malic enzyme and pyruvate kinase were less than 6 nanomoles/min/mg protein suggesting that they play a minor role in energy metabolism of the parasite. Phosphoenolpyruvate carboxykinase, pyruvate dehydrogenase, succinate dehydrogenase, NADP(+)-linked malic enzyme, NADH fumarate reductase, malate dehydrogenase, and alpha-ketoglutarate dehydrogenase and glycerol kinase on the other hand had specific activities greater than 60 nanomoles/min/mg protein. These enzyme activities could account for the production of pyruvate, acetate, succinate and glycerol. The results further show that the amount of glycerol produced was 35-48% of the combined total of pyruvate, acetate and succinate produced. It is apparent that some of the glycerol 3-phosphate produced in glycolysis in the presence of salicylhydroxamic acid is dephosphorylated to form glycerol while the rest is oxidised via cytochrome aa3 to form acetate, succinate and pyruvate.     

Relative Activities and Characteristics of Some Oxidative Respiratory Enzymes from Conidia of Verticillium albo-atrum

Conidia of Verticillium albo-atrum Reinke and Berthold, collected from shake cultures grown in Czapek broth, were sonified for 4 or 8 minutes or ground frozen in a mortar to obtain cell-free homogenates. These were assayed for certain enzymes associated with respiratory pathways. Malic dehydrogenase was the most active, glucose-6-P and NADH dehydrogenase were less active, NADH-cytochrome c reductase, NADPH dehydrogenase, and cytochrome oxidase were low in activity, and succinic dehydrogenase and succinic cytochrome c reductase were very low to negligible in activity. No NADH oxidase activity was detected.

With the exception of NADH-cytochrome c reductase and possibly succinic dehydrogenase and cytochrome c reductase, there was no evident increase in specific activity of the enzymes during germination. Some NADH-cytochrome c reductase and a small amount of succinic-dehydrogenase and cytochrome c reductase were associated with the particulate fraction from 105,000 × g centrifugation. The other enzymes, including cytochrome oxidase, almost completely remained in the supernatant fraction.

Menadione and vitamin K-S(II) markedly stimulated NADH-cytochrome c reductase activity in the supernatant fraction but had much less effect on NADPH-cytochrome c reductase in this fraction or on either of these enzyme systems in the particulate fraction. Electron transport inhibitors affected particulate NADH- and NADPH-cytochrome c reductase activity but had no effect on these in the supernatant fraction.

     

The oxidation of glucose by Acinetobacter calcoaceticus: interaction of the quinoprotein glucose dehydrogenase with the electron transport chain

The coupling of the quinoprotein glucose dehydrogenase to the electron transport chain has been investigated in Acinetobacter calcoaceticus. No evidence was obtained to support a previous suggestion that the soluble form of the dehydrogenase and the soluble cytochrome b associated with it are involved in the oxidation of glucose. Analysis of cytochrome content, and of reduction of cytochromes in membranes by substrates, and of sensitivity to cyanide indicated that glucose, succinate and NADH are all oxidized by way of the same b-type cytochrome(s) and cytochrome oxidases (cytochrome o and cytochrome d). Mixed inhibition studies [with KCN and hydroxyquinoline N-oxide (HQNO)] showed that the b-type cytochrome(s) formed a binary complex with the o-type oxidase and that there was thus no communication between the electron transport chains at the cytochrome level. Measurements of the reduction of ubiquinone-9 by glucose and NADH, and inhibitor studies using HQNO, indicated that the ubiquinone mediates electron transport from both the glucose and NADH dehydrogenases. In some conditions the quinone pool facilitated communication between the 'glucose oxidase' and 'NADH oxidase' electron transport chains, but in normal conditions these chains were kinetically distinct.