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PubMed1978

Reconstitution of escherichia coli succinoxidase from soluble components.

1. The membrane-bound succinoxidase of Escherichia coli was fractionated with deoxycholate into three soluble components, viz. succinate dehydrogenase.cytochrome b1 complex, cytochrome oxidase complex, and a factor identified as a phospholipid-containing component. 2. The dehydrogenase and cytochrome oxidase complexes were partially purified by filtration on Amicon membranes, Sepharose 4B chromatography, and sucrose gradient centrifugation. 3. Reconstitution of membranous succinoxidase, which catalyzes the oxidation of succinate by molecular oxygen by an integrated CN(-)-sensitive pathway, was achieved by mixing the soluble succinate dehydrogenase.cytochrome b1 complex with the soluble cytochrome oxidase complex in the presence of deoxycholate and then removing the detergent by gel filtration on Sephadex G-75. The phospholipid-containing factor stimulated the formation of succinoxidase by about 100% over that observed with the two complexes. 4. Isopycnic sucrose gradient centrifugation of succinate dehydrogenase.cytochrome b1 complex, cytochrome oxidase, and the reconstituted succinoxidase gave buoyant densities (p value) as 1.167, 1.229, and 1.194, respectively. 5. Electron microscopic evidence is presented for the vesicular nature of the reconstituted succinoxidase.

Cell Membrane↗
PubMed1987

Properties of bovine heart mitochondrial cytochrome b560.

A large-scale preparation of the two-subunit protein complex (QPs) that converts succinate dehydrogenase into succinate-ubiquinone reductase from cytochrome b-c1 particles is achieved by a procedure involving Triton X-100 solubilization and calcium phosphate column chromatography at different pH values. The isolated two-subunit QPs contains 25 nmol of cytochrome b560/mg of protein and is able to reconstitute with soluble succinate dehydrogenase to form a TTFA-sensitive succinate-ubiquinone reductase. The maximum reconstitutive activity is 100 mumol of succinate oxidized per min per mg of QPs protein at 23 degrees C. Although cytochrome b560 in isolated QPs is not succinate reducible and its dithionite reduced form is reactive to carbon monoxide, cytochrome b560 is shown to be physically associated with succinate dehydrogenase by the following observations. The dithionite reduced form of cytochrome b560 in isolated QPs has a symmetrical alpha-absorption peak, which upon reconstitution with succinate dehydrogenase becomes slightly broadened and shows a shoulder at around 553 nm, identical to that of cytochrome b560 in succinate-ubiquinone reductase. Upon addition of succinate dehydrogenase to QPs, about 50% of the reduced form of cytochrome b560 in the QPs becomes insensitive to carbon monoxide treatment. The redox potential of cytochrome b560 in QPs is -144 mV which is higher than that of cytochrome b560 in succinate-ubiquinone reductase (-185 mV). Upon addition of succinate dehydrogenase, the redox potential of about 46% of the cytochrome b560 in QPs preparation becomes identical to that of cytochrome b560 in succinate-ubiquinone reductase. Cytochrome b560 in the QPs preparation shows two epr signals, g = 3.07 and g = 2.92, whereas cytochrome b560 in succinate-ubiquinone reductase exhibits only one epr signal at g = 3.46. When QPs is reconstituted with succinate dehydrogenase to form succinate-ubiquinone reductase, the g = 3.46 epr signal reappears at the expense of the g = 3.07 signal. Based on epr measurement at liquid helium temperature, about 18% of the total cytochrome b in the isolated active succinate-cytochrome c reductase is cytochrome b560, indicating that cytochrome b560 is indeed a unique cytochrome b and not a denatured product of cytochrome b562 or b565.

Animals↗
PubMed1981

A new species of bound ubisemiquinone anion in QH2: cytochrome c oxidoreductase.

Using a combination of EPR and low temperature diffuse reflectance spectroscopy, a new species of semiquinone anion has been detected in QH2:cytochrome c oxidoreductase in submitochondrial particles under conditions of oxidant-induced extra reduction of cytochrome b. In contrast to the previously detected semiquinone anion, this new species is insensitive to antimycin but sensitive to treatment with 2,3-dimercaptopropanol and O2. The two species can easily be distinguished on the basis of their respective EPR properties since they differ in g-value, line width, and microwave power saturation behavior. It is concluded that the two species of semiquinone anion are bound to different domains on QH2:cytochrome c oxidoreductase. The existence of two different semiquinone anions in the enzyme strongly supports a mechanism of electron flow as proposed in the Q-cycle.

Animals↗
PubMed1982

Quantitative resolution of succinate-cytochrome c reductase into succinate-ubiquinone and ubiquinol-cytochrome c reductases.

A purified, active succinate-ubiquinone reductase was prepared from succinate-cytochrome c reductase without damage to ubiquinol-cytochrome c reductase by 1.1% Triton X-100 solubilization at pH 8.0, and calcium phosphate column chromatography in 50 mM Tris-succinate buffer, pH 8.0, containing 30 mM potassium phosphate. Succinate-ubiquinone reductase thus obtained contains ubiquinone and catalyzes thenoyltrifluoroacetone-sensitive oxidation of succinate by 2,6-dichlorophenolindophenol in the absence of exogenous mediator. Addition of ubiquinone enhanced the activity about 50%. Analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme contains four polypeptides. The high molecular weight polypeptide contaminants usually observed in the Complex II preparation obtained by the reported method were absent. The active succinate-ubiquinone reductase can reconstitute with the cytochrome b-c1III complex, or Complex III to form succinate-cytochrome c reductase in the absence of exogenous ubiquinone or with the resolved ubiquinol-cytochrome c reductase in the presence of ubiquinone and phospholipids. Under the proper conditions, all the original succinate-cytochrome c reductase was obtained, indicating that the resolution caused no damage to the protein, despite the removal of phospholipids and ubiquinone from the ubiquinol-cytochrome c reductase region.

Animals↗
PubMed1983

Properties of ATP tightly bound to catalytic sites of chloroplast ATP synthase.

Under steady state photophosphorylating conditions, each ATP synthase complex from spinach thylakoids contains, at a catalytic site, about one tightly bound ATP molecule that is rapidly labeled from medium 32Pi. The level of this bound [32P]ATP is markedly reduced upon de-energization of the spinach thylakoids. The reduction is biphasic, a rapid phase in which the [32P] ATP/synthase complex drops about 2-fold within 10 s, followed by a slow phase, kobs = 0.01/min. A decrease in the concentration of medium 32Pi to well below its apparent Km for photophosphorylation is required to decrease the amount of tightly bound ATP/synthase found just after de-energization and before the rapid phase of bound ATP disappearance. The [32P]ATP that remains bound after the rapid phase appears to be mostly at a catalytic site as demonstrated by a continued exchange of the oxygens of the bound ATP with water oxygens. This bound [32P]ATP does not exchange with medium Pi and is not removed by the presence of unlabeled ATP. The levels of tightly bound ADP and ATP arising from medium ADP were measured by a novel method based on use of [beta-32P]ADP. After photophosphorylation and within minutes after the rapid phase of bound ATP loss, the measured ratio of bound ADP to ATP was about 1.4 and the sum of bound ADP plus ATP was about 1/synthase. This ratio is smaller than that found about 1 h after de-energization. Hence, while ATP bound at catalytic sites disappears, bound ADP appears. The results suggest that during and after de-energization the bound ATP disappears from the catalytic site by hydrolysis to bound ADP and Pi with subsequent preferential release of Pi. These and related observations can be accommodated by the binding change mechanism for ATP synthase with participation of alternating catalytic sites and are consistent with a deactivated state arising from occupancy of one catalytic site on the synthase complex by an inhibitory ADP without presence of Pi.

ATP Synthetase Complexes↗
PubMed1995

Inhibition of succinate:ubiquinone reductase and decrease of ubiquinol in nephrotoxic cysteine S-conjugate-induced oxidative cell injury.

The role of complex II in the cellular protection against oxidative stress was investigated in freshly isolated rat renal proximal tubular cells (PTC) with the use of the nephrotoxin S-(1,2-dichlorovinyl)-L-cysteine (DCVC). DCVC caused oxidative stress in PTC as determined by flow cytometry with dihydrorhodamine-123; this fluorescent probe is readily oxidized by primary hydroperoxides such as those formed during lipid peroxidation. The oxidative stress could be prevented by inhibition of the beta-lyase-mediated formation and covalent binding to cellular macromolecules of reactive DCVC metabolites, with amino oxyacetic acid (AOA), or by the antioxidant N,N'-diphenyl-p-phenylenediamine. Both AOA and DPPD also prevented cell death. The DCVC-induced oxidative stress was associated with a decrease in the succinate:ubiquinone reductase (SQR) activity of complex II, whereas NADH:ubiquinone reductase activity of complex I remained unaffected. AOA prevented the effect on SQR activity, whereas N,N'-diphenyl-p-phenylenediamine did not. Inhibition of SQR activity with thenoyl trifluoracetone (TTFA) potentiated the DCVC-induced oxidative cell injury, suggesting the involvement of SQR activity in an antioxidant pathway. To investigate this in greater detail, PTC were treated with an inhibitor of cytochrome-c-oxidase, KCN, in a buffer containing glycine, which prevents cell death by KCN. Glycine did not affect cell death by DCVC. KCN prevented the DCVC-induced oxidative stress and cell death. KCN cytoprotection could be prevented by inhibition of SQR activity with oxaloacetate or TTFA, whereas inhibition of either complex I or III with rotenone and antimycin, respectively, did not prevent it. The effect of DCVC on complex II was associated with a decrease in the cellular amount of reduced ubiquinone (QH2); the KCN-mediated cytoprotection was related to a 60% increase of cellular QH2. Rotenone almost completely inhibited ubiquinone reduction even in the presence of KCN, whereas oxaloacetate in combination with KCN resulted in QH2 levels comparable to control. This suggests that the SQR activity by complex II rather than the cellular content of reduced ubiquinone (QH2) is important as a part of the cellular antioxidant machinery in the cyto-protection against oxidative stress.

Animals↗
PubMed1994

Protein ubiquinone interaction. Synthesis and biological properties of 5-alkyl ubiquinone derivatives.

For the investigation of the protein-ubiquinone interaction in the succinate-cytochrome c reductase region of the bovine heart mitochondrial electron transport chain, a series of 5-alkyl-substituted ubiquinone derivatives (5-R-Q0C10) were synthesized and characterized. Syntheses of 5-ethyl-Q0C10, 5-propyl-Q0C10, 5-isopropyl-Q0C10, and 5-butyl-Q0C10, were archived through radical coupling reactions between 2,3-dimethoxy-6-decyl-1,4-benzoquinone (5-H-Q0C10) and the corresponding alkanoyl peroxides. Although the spectral and redox properties of 5-R-Q0C10 are very similar to those of 5-methyl-2,3 dimethoxy-6-decyl-1,4-benzoquinone, the biological electron transfer efficiencies of these derivatives differ significantly. The reducibility of these derivatives by succinate, as measured with succinate-Q reductase and the oxidizability as measured by ubiquinol-cytochrome c reductase, decreased as the size of the substituents increased. 5-Ethyl-Q0C10 has about 50% of the activity of 5-methyl-2,3-dimethoxy-6-decyl-1,4-benzoquinone, whereas molecules with 5-alkyl groups of three or more carbon atoms are virtually inactive as electron acceptors for succinate-Q reductase. Reduced form of the derivative with no substituent at the 5-position, 5-H derivative is more effectively oxidized by ubiquinol-cytochrome c reductase than does the 5-methyl derivative, the native form. The oxidation of 5-H derivative is in a concentration-dependent manner at low concentrations but exhibits a substrate inhibition at higher concentrations. No such substrate inhibition is observed when other 5-substituted Q derivatives are used. 5-H derivative is a better electron acceptor for succinate-Q reductase than any other Q derivatives and does not show substrate inhibition, even at high concentrations. These results indicate that the binding environment of the benzoquinone ring in succinate-Q reductase is more specific than that of ubiquinol-cytochrome c reductase.

Alkylation↗
PubMed1997

Chemical preconditioning: a cytoprotective strategy.

Brief ischemic or hypoxic episodes may increase or decrease tolerance towards subsequent severe ischemia in heart and brain. A similar phenomenon is observed after mild chemical inhibition of oxidative phosphorylation--chemical preconditioning. We have shown that chemical preconditioning can be induced by chemical inhibition of mitochondrial complex I and mitochondrial complex II. With a time interval of three hours between chemical pretreatment and massive inhibition of oxidative phosphorylation, recovery of population spike amplitude in hippocampal region CA1 after stimulation of the Schaffer collaterals was 31 +/- 9% in controls, 98 +/- 14% after i.p. treatment with 1 mg/kg body weight haloperidol, an inhibitor of mitochondrial complex I and 90 +/- 7% with pretreatment with 3-np, an inhibitor of mitochondrial complex II. Activation of ATP regulated potassium channels partakes in mediating the preconditioning effect. We conclude that chemical preconditioning is a practical prophylactic pharmacologic strategy to increase hypoxic tolerance.

Animals↗
PubMed1998

Dedifferentiated cardiomyocytes from chronic hibernating myocardium are ischemia-tolerant.

Left ventricular biopsies from 21 patients with clinically diagnosed chronic hibernating myocardium (CHM) were examined by light- and electron microscopy. A mean of 27% of cardiomyocytes were structurally altered and were characterized as hibernating, because of reduced amount of myofibrils and increased glycogen content. Electron microscopy of these cells showed reduction of T-tubules and numerous small mitochondria, but few changes associated with degeneration, acute ischemia or apoptosis. The structural changes found in CHM are reminiscent of dedifferentiation rather than degeneration. The expression patterns of some structural proteins show resemblance with those in embryonic cardiomyocytes. Histochemically, mitochondrial NADH-oxidase and proton translocating ATPase activities were absent, whereas cytochrome c activity was present. Intracellular calcium distribution indicated normally bound sarcolemmal calcium and absence of excess mitochondrial calcium accumulation. Nuclear chromatin ranged from normal to dispersed with only a few nuclei that were clumped. These results suggest that cardiomyocytes from human CHM hearts are structurally altered, but viable, and lack morphologic and cytochemical characteristics suggestive of apoptosis or acute ischemia.

Apoptosis↗
PubMed1980

[New ultrasonic method for studying the composition and properties of multienzyme systems: enzymes of the cellulase complex].

A new ultrasonic method for determination of the composition and properties of individual components of multienzyme systems without their resolution has been developed. The method is based on a determination of the pH-profiles of the first order rate constants for inactivation of the enzymatic components in a complex by ultrasonic cavitation. The method was used for studying a cellulase complex from Geotrichum candidum. It was shown that the cellulase complex contains at least four cellulolytic enzymes, i. e. endoglucanase, exoglucosidase, cellobiase and aryl-beta-glucosidase, which differ in their pK values for the ionogenic groups controlling the pH-profiles of ultrasonic inactivation and in the inactivation rate constants at the plateau of a pH-rate profile.

Cellulase↗