Electron Transport Particles Research Articles

Action of halothane upon mitochondrial respiration

The inhibitory action of halothane upon respiration was studied with rat liver mitochondria (RLM 3), beef heart mitochondria (HBHM), and electron-transport particles (ETP). With intact mitochondrial preparations the oxidation of NADH-linked substrates but not of succinate was markedly suppressed by low concentrations of halothane (<2 m m as determined by gas-liquid chromatography). This inhibitory action of halothane was completely reversible. In contrast, a number of other mitochondrial processes were found to be sensitive in an irreversible manner at higher concentrations of the anesthetic. Likewise, the oxidation of added NADH by HBHM, ETP, and detergent-disrupted RLM was found to be sensitive in a reversible manner to low concentrations of halothane. The energy-dependent transfer of electrons from succinate to NAD by ETP H was also sensitive to halothane. On the other hand, the NADH-ferricyanide reductase and the succinic oxidase activities of ETP and the NADH-cytochrome c reductase activity of microsomes were all insensitive to halothane. The site of inhibition by halothane appears to be in the vicinity of the rotenone-sensitive site of complex I (NADH-CoQ reductase). A number of other general anesthetics inhibited respiration at or near the same site as halothane.

The Influence of Seasonal Variation on Oxidative Phosphorylation in Sub-mitochondrial Particles Prepared from Jerusalem Artichoke Tubers

A method for the preparation of sub-mitochondrial particles from Jerusalem artichoke tubers by sonication is described. The particles carried out a rapid oxidation of NADH, succinate, and ascorbate saturated with N',N',N, A-tetramethylphenylenediamine (TMPD). The difference spectrum (dithionite reduced minus oxidized) of sub-mitochondrial particles was similar to that of whole mitochondria. In the autumn phosphorylation accompanied electron transport only when supernatant from the preparation procedure was added to the sub mitochondrial particles in the presence of magnesium. In the winter and spring there was a decrease in the effect of supernatant on phosphorylation, and the sub-mitochondrial particles alone synthesized ATP during substrate oxidation. It is suggested that the reconstitution of phosphorylation in Jerusalem artichoke sub-mitochondrial particles is essentially similar to that observed in beef heart electron transport particles, but that phosphorylation in the former is subject to seasonal variations.

Activation energies for the ATP-driven reversal of oxidative phosphorylation in submitochondrial particles

The participation of energy transfer Factor B at the second phosphorylation site between cytochromes b and c 1 in the respiratory chain was tested spectroscopically at the wavelength pair 562–575 mμ with ascorbate plus N, N, N′, N′-tetramethyl- p-phenylenediamine (TMPD) as substrate and ATP as energy source for the reversal of oxidative phosphorylation. At temperatures above 30°, Factor B produced a distinct stimulation of the activity of ammonia particles but did not alter the extent of cytochrome b reduction. Below 30° no effect of Factor B could be observed. A similar though not completely matching biphasic thermal effect was also seen in the reversal of oxidative phosphorylation between NADH dehydrogenase and cytochrome b (site 1) promoted by Factor A or B with the urea and ammonia-EDTA particles, respectively. A phosphorylating electron transport particle from heavy mitochondria (ETP H) showed the same biphasic thermal respose in the ATP-driven NAD + reduction as well as in its oligomycin-sensitive ATPase activity without the addition of any factors. In all the above cases, there was a break in the Arrhenius plot around 30° except for the reversal of oxidative phosphorylation at site 1 in ETP H. The activation energies and their changes on supplementation with the energy transfer factors differed for the particles. Factor B had no effect on the activation energy of ammonia particles in the reversal of site 1 or 2 but simply increased the rate of the reversal. The effect of oligomycin at low levels in the reversal of site 1 phosphorylation with ammonia particles was similar to the effect of Factor B. It is concluded, in agreement with previous reports, that oligomycin and Factor B act in the same region of the energy transfer reactions. Factor A strikingly decreased the activation energy for the reversal of phosphorylation at site 1 below 31° in urea particles from 43.8 to 24.3 kcal/mole, supporting a structural role for the factor in stimulating oxidative phosphorylation. Above 31° the change was from 27.5 to 20.5 kcal/mole. Omission of dithiothreitol or addition of low levels of p-chloromercuriphenylsulfonate (PCMS) produced a decrease in the rate of reversal of site 1 phosphorylation in ETP H. Concomitant with this decrease a break was induced in the Arrhenius plot at about 28°. The activation energy above the break was between 11.8 and 12.8 kcal/mole, similar to the value for Factor B-supplemented ammonia particles in the same temperature range. Below the transition temperature, the activation energy was 24.6–26.2 kcal/mole. It is concluded from these studies that reconstitution of oxidative phosphorylation occurs best at temperatures between 30 and 43° possibly due to easier reassembly of the membrane structure at the higher temperatures.

Effect of proteolytic and lipolytic enzymes on the electron transport particle fraction of rhodospirillum rubrum

Digestion of the electron transport particle fraction of Rhodospirillum rubrum with the proteases chymotrypsin, trypsin, subtilisin and pronase resulted in a release of protein from the membranal system. The solubilization was, however, limited to only 15 - 18 percent of the total protein, being the same with each of the four proteases. The enzymes catalyzing electron transfer which are located on these particles were not solubilized by the proteolytic digestion but were found to become differently inactivated already with low protease concentrations. The proteolytic attack revealed differences in the specificity of proteolysis between chymotrypsin and the other proteases

The relation of phospholipid and membrane structure in mitochondrial electron transport particles

The relation of phospholipid and membrane structure in mitochondrial electron transport particles

The relation of phospholipid and membrane-bound ATPase in mitochondrial electron transport particles

The relation of phospholipid and membrane-bound ATPase in mitochondrial electron transport particles

The effect of trypsin and heat treatment on oxidative phosphorylation in Mycobacterium phlei

The electron transport particles from Mycobacterium phlei contain particle-bound coupling factors and require the addition of soluble coupling factors for restoration of phosphorylation. However, exposure of the particles to heat (50°) or treatment with trypsin increased the P/O ratio with succinate or with NAD +-linked substrates. In addition, the heat treated particles no longer required the soluble coupling factors for phosphorylation, but the trypsin-treated particles required this addition for maximal levels of phosphorylation. A combination of treatment with trypsin and heat resulted in higher levels of phosphorylation than either treatment alone. The phosphorylation observed in both cases was sensitive to uncoupling agents.

Spin Label Studies of Hemoproteins: I. CYTOCHROME c

Abstract Bakers' yeast cytochrome c was spin labeled with N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)-bromoacetamide at the cysteine residue. The optical properties of the spin-labeled cytochrome c were similar to those of the native protein. The electron-transfer activity of the modified cytochrome c in the mitochondrial NADH oxidase system was 100%. The electron paramagnetic resonance spectrum of either ferri- or ferrocytochrome c indicated weak immobilization of the spin label. Upon mixing with cytochrome c peroxidase, the electron paramagnetic resonance spectrum of the labeled cytochrome c was only slightly broadened. The incorporation of the spin-labeled cytochrome c into electron transport particles resulted in a greater immobilization of the label. The latter interaction was quite sensitive to the ionic strength of the suspending medium when the cytochrome c was bound to the outside of the particles. On the other hand, the spin-labeled cytochrome c either in solution or loaded to the inside of the particles was insensitive to the ionic strength.

Effects of heat treatment of electron-transport particles on bacterial oxidative phosphorylation.

The electron-transport particles from Mycobacterium phlei exhibit low levels of phosphorylation unless supplemented with soluble coupling proteins. Heat treatment of the electron transport particles for 15 min at 50 degrees C was found to result in a slight loss of oxidation and an activation of phosphorylation with NADH as substrate, while with succinate as substrate both activities increased. The heat-activated particles do not require the addition of soluble coupling factors and their level of oxidative phosphorylation is similar to that of the regular particles supplemented with the soluble coupling factors. In contrast to the lack of a requirement for the soluble coupling factors after heat activation, the heat-treated electron-transport particles require the presence of a particulate-bound coupling factor for phosphorylation. The heat-activated system, like the untreated system, was found to be sensitive to uncoupling agents.

Electron transport particles released upon lysis of spheroplasts of Escherichia coli B by Brij 58

The non-ionic detergent, Brij 58, has been shown to specifically lyse the cytoplasmic membrane of Escherichia coli. This communication examines the electron transport system in membrane fractions prepared from such lysates and compares this system to those prepared by mechanical procedures. 1. 1. The particulate fraction contained all of the respiratory carriers demonstrable in whole cells in essentially the same ratios although enriched by 3–5-fold over their concentration in whole cells. 2. 2. Succinate, formate, and NADH but not glutamate, malate, or dihydroorotate were actively oxidized by the particulate fraction. 3. 3. With the exception of the formate oxidase system which appeared to utilize a system bypassing flavoprotein and cytochrome b 1, the other enzymatic activities appeared to function primarily through normal electron transport routes. 4. 4. The NADH oxidase and succinoxidase systems were sensitive to inhibition by 2- n-heptyl-4-hydroxyquinoline- N-oxide and antimycin A. The results suggest that these inhibitors function at the level of cytochrome b 1. 5. 5. All three activities were sensitive to complete inhibition by CN −. 6. 6. The results obtained from inhibitor studies coupled with the results obtained from examination of steady state, anaerobic, and chemical reduction of the respiratory pigments permitted a scheme for electron flow to be proposed.

Cytochromes from Mycobacterium phlei: I. Isolation and spectral properties of a mixture of cytochromes ( a + a3) ( o)

A complex containing cytochromes a + a 3 and cytochrome o was isolated from Mycobacterium phlei by solubilization with Triton X-100 and subsequent digestion with trypsin. Further purification was achieved by fractionation with ammonium sulfate. The spectra of the oxidized, the reduced, the pyridine hemochromogens, the carbon monoxide, and the cyanide complexes of cytochromes a + a 3 and cytochrome o were presented. The results obtained with cytochromes a + a 3 were similar to those described for mammalian systems. Over a pH range of 6.0 to 8.0 both cytochrome a 3 and o were reducible by ascorbate- N,N,N′,N′-tetramethylphenylenediamine or by NADH plus a catalytic amount of M. phlei electron transport particles. However, cytochrome o was reducible by dithionite only when carbon monoxide or cyanide treatment preceded chemical reduction. The chemical reduction of cytochrome a 3 was found to be independent of the order of addition of carbon monoxide or cyanide.

Lipid oxidation in biological membranes: I. Lipid oxidation in submitochondrial particles and microsomes induced by chaotropic agents

Chaotropic agents (SCN −, guanidine-HCl, ClO 4 −, I −, Br −, NO 3 −, urea) destabilize the native structure of Submitochondrial particles and microsomes and render these systems susceptible to lipid oxidation by molecular oxygen. The initial rate of lipid oxidation varies with the concentration and the potency of the chaotropic agent used. The order of decreasing potency of chaotropic agents in inducing lipid oxidation in submitochondrial particles is essentially the same as given above. In electron-transport particles and complexes I, III, and IV of the respiratory chain, lipid oxidation as induced by chaotropic agents shows a rapid initial rate followed by a slower rate. Preparations with a high cytochrome content are capable of lipid oxidation when incubated at 30 ° in the absence of any chaotropic agent. This uninduced reaction is particularly significant in preparations of cytochrome oxidase, which are capable of oxygen consumption at a capacity of 1.76 μmoles O 2/mg of protein. Lipid oxidation in both submitochondrial particles and microsomes results in the production of malondialdehyde. The production of this compound is much greater at the initial phases of lipid oxidation. Substrates impede lipid oxidation in electron-transport particles, and antimycin A inhibits lipid oxidation in complex III. α-Tocopherol, Tiron, and bathophenanthroline sulfonate also inhibit lipid oxidation. Results are discussed in relation to the structure, function, and lipid composition of Submitochondrial particles and microsomes.

Studies on the Respiratory Chain-linked Reduced Nicotinamide Adenine Dinucleotide Dehydrogenase: XVII. REACTION SITES OF PIERICIDIN A AND ROTENONE

Abstract An improved estimate from Scatchard plots of the number of specific binding sites of the electron transport particle for 14C-piericidin A gives a value of 2 moles of inhibitor bound per mole of calculated NADH dehydrogenase content. The affinity of these two specific binding sites for piericidin A is very similar, since the binding curve is a smooth hyperbola and the Scatchard plot is linear. The two sites may be distinguished, however, by their unequal contribution to the inhibition of NADH oxidation. This is shown by the sigmoidicity of the curves relating inhibition of NADH oxidase, NADH-coenzyme Q reductase, and energy-linked NAD reductase activities to piericidin concentration. The inhibition of NADH oxidase and energy-linked NAD reductase activities but not of NADH-coenzyme Q reductase activity are partially reversed by washing of inhibited particles with bovine serum albumin. Hence both specifically and unspecifically bound piericidin contribute to the inhibition of the first two activities while only specifically bound inhibitor is involved in the block of external coenzyme Q reduction. The titration curve for inhibition of NADH oxidase by rotenone is more complex, apparently because of the contribution of inhibition at unspecific sites. The number of specific binding sites for piericidin may be reduced to near 1 per mole of NADH dehydrogenase by treatment of the electron transport particle with mercurials and by dissociation into the component complexes with bile salts. The results are discussed in terms of the location of the binding and inhibition sites of rotenone and piericidin in the respiratory chain and of the possible participation of NADH dehydrogenase itself and other membrane components in the binding of these inhibitors.

Reaction sites of rotenone, piericidin A, and amytal in relation to the nonheme iron components of NADH dehydrogenase.

The locus of inhibition of nicotinamide adenine dinucleotide, reduced form (NADH) oxidation in mitochondria by rotenone, piercidin A, and barbiturates is considered in the light of available information. Most lines of evidence indicate that the point of inhibition is on the O(2) side of NADH dehydrogenase. Kinetic experiments on the substrate-induced appearance of the electron paramagnetic resonance signal at g = 1.94 in membrane preparations (ETP) reveal that these inhibitors do not interfere with the reduction of the electron paramagnetic resonance detectable iron by NADH. Our spectrophotometric studies on complex I give no evidence for absorbance differences between untreated and rotenone or piericidin inhibited preparations, which can be attributed to nonheme iron. Whatever changes were observed appear to be due to cytochromes. These experiments, therefore, do not support the idea that in inhibited preparations electron transport is interrupted between the flavin and nonheme iron components of NADH dehydrogenase. The specific binding of rotenone and piericidin seems to involve both lipid and protein. The possibility that NADH dehydrogenase participates in the binding is suggested by the apparent stoichiometric relation between specific binding site titer and NADH dehydrogenase content and the profound effect of mersalyl inhibition of the enzyme on piericidin binding capacity.ETP, electron transport particle.

Oxidative phosphorylation in fractionated bacterial systems: XLII. The effect of coupling factors on urea-treated particles from M. phlei

The electron transport particles from Mycobacterium phlei are capable of coupling phosphorylation to the oxidation of succinate or generated NADH. Treatment of the particles with urea resulted in the loss of the phosphorylative activity without any effect on the oxidation of either substrate. Addition of a factor (BCF 1) released by urea treatment of particles to the urea-treated particles restored phosphorylation to the original level with succinate as the electron donor. This factor, however, was not effective when exogenous NADH (1) or ascorbate-TPD were used as substrates. The addition of a combination of the factor released by urea treatment and the soluble coupling factor (BCF 3) resulted in an increase in the level of phosphorylation. In contrast, with ascorbate and TPD as the electron donors only BCF 3 was capable of restoring phosphorylation. The restored phosphorylation was found to be sensitive to uncoupling agents to the same degree as that observed with these agents in the unresolved system. Antibodies prepared to the BCF 3 fraction were found to inhibit the phosphorylation elicited by this factor and had no effect on the phosphorylation restored by the other coupling factors (BCF 1 and 2). Evidence has been obtained which indicates that the factor released by urea treatment of the particles may participate in the reaction(s) common to both the succinate and NAD +-linked pathways and that it is different from the soluble coupling factors.

Oxidative phosphorylation in fractionated bacterial systems XLIII. Coupling factors associated with the NAD +-linked electron transport pathway

Oxidative phosphorylation with the cell-free system from Mycobacterium phlei is dependent on the presence of both the particulate and soluble fractions. While the particles contain the bulk of the respiratory components, the soluble fraction contains a number of dehydrogenases which are necessary to initiate electron transport and protein components which are necessary for the coupling process. The supernatant components necessary for restoration of phosphorylation were resolved into two different coupling factors which appear to operate at different phosphorylative sites. The purified coupling factors appear to be protein in nature. The phosphorylation restored on addition of the purified coupling factors to the electron transport particles was found to be sensitive to uncoupling agents. The purified coupling factors were found to contain some ATPase, adenylate kinase, 14C-ADP-ATP and 32P i-ATP exchange activities. Antibodies prepared to either of the coupling factors was found to be specific for the coupling protein to which it was prepared. In addition, the antibodies were found to inhibit the phosphorylation elicited by that coupling factor. An additional coupling factor was found to be bound to the electron transport particles. The particulate bound factor was released from the particles by treatment with 1.0 m urea. The level of phosphorylation was increased by a combination of the different coupling factors. The coupling factors had no significant effect on the level of oxidation.

Interchangeability of coupling factors from bacterial and mammalian origin

Bacterial and mammalian preparations capable of oxidative phosphorylation can be separated into a particulate fraction containing the electron transport chains and a soluble fraction containing coupling proteins necessary for restoration of phosphorylation. The addition of coupling factors of bacterial origin to submitochondrial particles was found to replace the requirement for mammalian coupling factors for restoration of phosphorylation. Coupling factors obtained from light beef heart mitochondria were found to restore phosphorylation to the electron transport particles of Mycobacterium phlei . The level of phosphorylation restored by the heterologus coupling factors was found to be lower than that restored by the native coupling factor; however, the phosphorylation restored by either coupling factor was found to be sensitive to uncoupling agents.

Studies with Triton X-100 treated electron transport particles from Rhodospirillum rubrum.

The effects of Triton X-100 on the membranal fraction containing the electron transport system in R. rubrum were studied. 1. The NADH oxidase system is very sensitive to treatment with Triton X-100. An inactivation of 50% is already found with a detergent/membrane protein ratio of 0.012(w/w). 2. NADH-cytochrome c-reductase also becomes very rapidly inactivated. 3. The activity of succinate-cytochrome c-reductase is stimulated by low and inactivated by higher concentrations of Triton X-100. The increase in activity is appr. 200%. 4. NADH dehydrogenase with menadione as electron acceptor is partly inactivated by low concentrations of Triton. Higher concentrations of the detergent will again lead to a stimulation of the activity. In the presence of these higher detergent concentrations the kinetics of the reaction of the enzyme are altered, i.e. an increase of up to 300% of the reaction rate is found during assay. The low initial rate is slowly increased with the time the samples containing Triton are kept at 0° C. Elevated temperatures (20° C) or dilution of the sample prior to assay will cause the initial rate to rise immediately. 5. Succinate dehydrogenase, NADH dehydrogenase with DCPIP, ferricyanide and also the activity with menadione are very stable against treatment with Triton. A detergent/membrane protein ratio of 12.5 will result in only slight inactivation of these activities.

Alpha-hydroxyglutarate oxidoreductase of Pseudomonas putida.

Oxidation of d-alpha-hydroxyglutarate to alpha-ketoglutarate is catalyzed by d-alpha-hydroxyglutarate oxidoreductase, an inducible membrane-bound enzyme of the electron transport particle [ETP; a comminuted cytoplasmic membrane preparation with enzymic properties and chemical composition resembling beef heart mitochondrial ETP (1)] of Pseudomonas putida P2 (P2-ETP). Treatment of P2-ETP with a nonionic detergent yields a preparation with the sedimentation characteristics of a soluble enzyme, but which retains an intact electron transport chain. Oxygen acts solely as a terminal electron acceptor and may be replaced by ferricyanide, 2,6-dichlorophenol indophenol, or mammalian cytochrome c. The oxidoreductase is specific for the d-isomer (K(m) = 4.0 x 10(-4)m for dl-alpha-hydroxyglutarate) and is distinct both from l- and d-malate dehydrogenases. Spectral studies suggest that the carrier sequence is substrate --> flavine or nonheme iron --> cyt b --> [cyt c] --> oxygen.

Extraparticulate chain interaction between different electron transport particles.

Interaction or cross-linking between the respiratory chains of the electron transport particles of bacterial origin occurs with a mixture of active and inactive particles. Interaction between bacterial particles and liver sub-mitochondrial particles also occurs. Irradiation of the bacterial particles at 360 nanometers resulted in the destruction of quinone and consequent loss of ability of reduced nicotinamide adenine, dinucleotides to reduce cytochromes b, c(1), c, and a plus a(3). A mixture of both irradiated and untreated particles in the presence of the reduced dinucleotide resulted in the reduction of cytochromes c and a plus a(3), in an amount equivalent to the total concentration of these cytochromes in both types of particles. In contrast, the amount of cytochrome b reduced was equivalent to half the particle concentration or to that observed with the active particles alone. The rate of reduction of cytochromes c and a plus a(3) with the mixture of particles was similar to that with the active particles alone. The interaction or cross-linking between the particulate respiratory chains of bacteria or of bacterial and mammalian systems occurs after cytochrome b and before or at cytochrome c.