Respiratory Electron Transport Systems of Aquatic Fungi. I. Leptomitus lacteus and Apodachlya punctata

The electron transport systems of 2 species of aquatic fungi, Leptomitus lacteus and A podachlya punctata, contained cytochrome a-a₃ (605 mμ), 2 b type cytochromes (564 and 557 mμ), c type cytochrome (551 mμ), and flavoprotein, but they appeared to lack cytochrome c₁. Reduced-minus-oxidized difference spectra and difference spectra in the presence of antimycin A or cyanide were used to characterize these systems. Studies with the electron microscope revealed that hyphae of Leptomitus lacteus contained numerous, conspicuous mitochondria with tubular cristae.     

Dihydroorotate-dependent superoxide production in rat brain and liver. A function of the primary dehydrogenase.

Dihydroorotate dehydrogenase in rat brain mitochondria is capable of producing superoxide. The presence of a superoxide dismutase activity in brain mitochondria, similar to that found in mitochondria from chicken liver, suggests that production of superoxide may occur in vivo. Formation of superoxide is not dependent upon reduction of cytochrome b, rather, superoxide production is competitive with cytochrome b reduction. Phenazine methosulfate apparently competes with both oxygen (superoxide production) and cytochrome b as an electron carrier but does not enhance reduction of dichlorophenolindophenol or cytochrome c.     

The NT-26 cytochrome c552 and its role in arsenite oxidation

Arsenite oxidation by the facultative chemolithoautotroph NT-26 involves a periplasmic arsenite oxidase. This enzyme is the first component of an electron transport chain which leads to reduction of oxygen to water and the generation of ATP. Involved in this pathway is a periplasmic c-type cytochrome that can act as an electron acceptor to the arsenite oxidase. We identified the gene that encodes this protein downstream of the arsenite oxidase genes (aroBA). This protein, a cytochrome c₅₅₂, is similar to a number of c-type cytochromes from the α-Proteobacteria and mitochondria. It was therefore not surprising that horse heart cytochrome c could also serve, in vitro, as an alternative electron acceptor for the arsenite oxidase. Purification and characterisation of the c₅₅₂ revealed the presence of a single heme per protein and that the heme redox potential is similar to that of mitochondrial c-type cytochromes. Expression studies revealed that synthesis of the cytochrome c gene was not dependent on arsenite as was found to be the case for expression of aroBA.     

The respiratory chain components of higher plant mitochondria

Tightly coupled mitochondria have been prepared from a variety of plant sources: white potato (Solanum tuberosum), Jerusalem artichoke (Heliantus tuberosus), cauliflower buds (Brassica oleracea), and mung bean hypocotyls (Phaseolus aureus). Mitochondria with no appreciable coupling were also prepared from skunk cabbage spadices (Symplocarpus foetidus).

Room temperature difference spectra show that these mitochondria are very similar in the qualitative and quantitative composition of their electron carriers. The different cytochromes are present in the amounts of 0.1 to 0.3 mμmole per mg of mitochondrial protein. The molar ratios of the different electron carriers are, on the average: 0.7:0.7:1.0:3 to 4:10 to 15 respectively for cytochrome aa₃, cytochromes b, cytochromes c, flavoproteins, and pyridine nucleotides.

From low temperature difference spectra carried out under particular experimental conditions, it can be deduced that these mitochondria contain 3 b cytochromes whose α bands are located at 552, 557, and 561 mμ, and 2 c cytochromes, one of which, a c₁-like cytochrome, is firmly bound to the mitochondrial membrane. Cytochrome oxidase can be optically resolved into its 2 components a and a₃.

For all kinds of mitochondria, the rates of oxidation of succinate are similar as well as the turnover of cytochrome oxidase (50-70 sec⁻¹), regardless of the metabolic activities of the tissues. The number of mitochondria per cell appears to be the controlling factor of the intensity of tissue respiration.

     

Isolating the segment of the mitochondrial electron transport chain responsible for mitochondrial damage during cardiac ischemia

Ischemia damages the mitochondrial electron transport chain (ETC), mediated in part by damage generated by the mitochondria themselves. Mitochondrial damage resulting from ischemia, in turn, leads to cardiac injury during reperfusion. The goal of the present study was to localize the segment of the ETC that produces the ischemic mitochondrial damage. We tested if blockade of the proximal ETC at complex I differed from blockade distal in the chain at cytochrome oxidase. Isolated rabbit hearts were perfused for 15 min followed by 30 min stop-flow ischemia at 37 °C. Amobarbital (2.5 mM) or azide (5 mM) was used to block proximal (complex I) or distal (cytochrome oxidase) sites in the ETC. Time control hearts were buffer-perfused for 45 min. Subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) were isolated. Ischemia decreased cytochrome c content in SSM but not in IFM compared to time control. Blockade of electron transport at complex I preserved the cytochrome c content in SSM. In contrast, blockade of electron transport at cytochrome oxidase with azide did not retain cytochrome c in SSM during ischemia. Since blockade of electron transport at complex III also prevented cytochrome c loss during ischemia, the specific site that elicits mitochondrial damage during ischemia is likely located in the segment between complex III and cytochrome oxidase.     

The electron transport systems of Fasciola hepatica mitochondria.

The electron transport systems of Fasciola hepatica mitochondria were investigated spectrophotometrically at room temperature and at −196°. The mitochondria were found to contain substrate reducible a-, b- and c-type cytochromes. All of the cytochrome components of the classical mammalian type of respiratory chain were present, although the concentration of cytochromes aa₃ was low. In addition to the mammalian type of respiratory chain, the Fasciola mitochondria contained a substrate reducible b-type cytochrome component (557 nm) which included a CO reactive o-type cytochrome. The results suggest that F. hepatica mitochondria contain a branched electron transport system including a mammalian type of chain and involving two terminal oxidases and at least two b-type cytochromes.     

The Respiratory Chain of Plant Mitochondria: XVI. Interaction of Cytochrome b(562) with the Respiratory Chain of Coupled and Uncoupled Mung Bean Mitochondria: Evidence for Its Exclusion from the Main Sequence of the Chain

Cytochromes b₅₅₃, b₅₅₇, and b₅₆₂ of mung bean (Phaseolus aureus) mitochondria become partially reduced with endogenous substrate on addition of antimycin A to the aerobic mitochondrial suspension. Addition of ATP causes partial reoxidation of the three cytochromes. This partial oxidation by ATP is inhibited by oligomycin and reversed by uncoupler. Ubiquinone does not appear to act as electron acceptor for the oxidation reaction, but a nonfluorescent flavoprotein, or possibly ironsulfur protein, component does appear to act as acceptor. This is consistent with reverse electron transport driven by ATP across the first site of energy conservation of the respiratory chain. Endogenous pyridine nucleotide and the fluorescent flavoprotein with E_m7.2 = −155mv (midpoint potential at pH 7.2, referred to normal hydrogen electrode) in uncoupled mitochondria become reduced in anaerobiosis attained by oxidation of succinate in the absence of respiratory inhibitors of the cytochrome chain, provided that Pi and ATP are present. Under these same conditions, cytochrome b₅₅₇ is completely reduced but cytochrome b₅₆₂ remains nearly completely oxidized. There is no equilibration across the first site of energy conservation between the carriers on the low potential side and cytochrome b₅₆₂ with E_m7.2 = −77mv on the high potential side. It is concluded that cytochrome b₅₆₂ is not a part of the main sequence of electron transport carriers of the mitochondrial respiratory chain of plants; it can participate in redox reactions with the respiratory chain in coupled mitochondria but not in uncoupled mitochondria unless antimycin A is present.     

Intermembrane electron transport in the absence of added water-soluble carriers

Electron transport from untreated to mersalyzed microsomal vesicles at the level of NADH-cytochrome b₅ reductase or cytochrome b₅ has been demonstrated in the absence of added water-soluble electron carriers. A similar effect was shown in the systems “intact mitochondria — mersalyzed microsomes” and “mersalyzed mitochondria— untreated microsomes”. No measurable electron transport between intact and mersalyzed particles of inner mitochondrial membrane was found. The obtained data suggest that the capability to carry out intermembrane electron transfer is specific for NADH-cytochrome b₅ reductase and/or cytochrome b₅, localized in microsomal and outer mitochondrial membranes.     

Properties of rat liver mitochondria with intermembrane Cytochrome c.

When rat liver mitochondria were suspended in 0.15 m KCl, the cytochrome c appeared to be solubilized from the binding site on the outside of the inner membrane and trapped in the intermembrane space. When the outer membrane of these mitochondria was disrupted with digitonin at a digitonin concentration of 0.15 mg/mg of protein, the solubilized cytochrome c could be released from mitochondria along with adenylate kinase. When mitochondria were suspended in 0.15 m KCl instead of 0.33 m sucrose, the $\text{ADP}\text{O}$ ratio observed with succinate, β-hydroxybutyrate, malate + pyruvate or glutamate as substrates was little affected. A number of cycles of State 4-State 3-State 4 with ADP was observed. The respiratory control ratios, however, were decreased, particularly when glutamate was used as the substrate. Cytochrome c oxidase activity was also decreased to 55% when assayed using ascorbate + N,N,N′,N′-tetramethyl-p-phenylene-diamine (TMPD) as substrates. Suspension of mitochondria in 0.15 m KCl resulted in an enhancement of the very low NADH oxidation by intact mitochondria and a twofold enhancement of sulfite oxidation. Trapped cytochrome c in outer membrane vesicles prepared from untreated and trypsin-treated intact mitochondria was found to be readily reduced by NADH and suggests that some cytochrome b₅ is located on the inner surface of the outer membrane. The enhanced NADH oxidase could therefore reflect the ability of cytochrome c to mediate intermembrane electron transport. The enhanced sulfite oxidase activity was sensitive to cyanide inhibition and coupled to oxidative phosphorylation ($\text{ADP}\text{O}\text{< 1}$) unlike the activity of mitochondria in sucrose medium. These results suggest that free cytochrome c in the intermembrane space can mediate electron transfer between the sulfite oxidase and the inner membrane.     

The parkinsonian neurotoxin MPP+ opens the mitochondrial permeability transition pore and releases cytochrome c in isolated mitochondria via an oxidative mechanism

The mitochondrial transition pore (MTP) is implicated as a mediator of cell injury and death in many situations. The MTP opens in response to stimuli including reactive oxygen species and inhibition of the electron transport chain. Sporadic Parkinson’s disease (PD) is characterized by oxidative stress and specifically involves a defect in complex I of the electron transport chain. To explore the possible involvement of the MTP in PD models, we tested the effects of the complex I inhibitor and apoptosis-inducing toxin N-methyl-4-phenylpyridinium (MPP⁺) on cyclosporin A (CsA)-sensitive mitochondrial swelling and release of cytochrome c. In the presence of Ca²⁺ and P_i, MPP⁺ induced a permeability transition in both liver and brain mitochondria. MPP⁺ also caused release of cytochrome c from liver mitochondria. Rotenone, a classic non-competitive complex I inhibitor, completely inhibited MPP⁺-induced swelling and release of cytochrome c. The MPP⁺-induced permeability transition was synergistic with nitric oxide and the adenine nucleotide translocator inhibitor atractyloside, and additive with phenyl arsine oxide cross-linking of dithiol residues. MPP⁺-induced pore opening and cytochrome c release were blocked by CsA, the Ca²⁺ uniporter inhibitor ruthenium red, the hydrophobic disulfide reagent N-ethylmaleimide, butacaine, and the free radical scavenging enzymes catalase and superoxide dismutase. MPP⁺ neurotoxicity may derive from not only its inhibition of complex I and consequent ATP depletion, but also from its ability to open the MTP and to release mitochondrial factors including Ca²⁺ and cytochrome c known to be involved in apoptosis.     

Effect of dietary lysine on performance and expression of electron transport chain genes in the pectoralis major muscle of broilers

The aim of this study was to evaluate the effect of dietary lysine on performance, protein deposition and respiratory chain gene expression in male broilers. A total of 252 Cobb 500 broilers were distributed, in a completely randomized design, into four treatments with seven replicates of nine birds per experimental unit. Experimental treatments consisted of diets based on corn and soybean meal, with four levels of digestible lysine: 1.016%, 1.099%, 1.182% and 1.265%. The increase in the level of digestible lysine in the diet provided higher weight gains, feed efficiency and body protein deposition. Birds fed the lowest level of dietary lysine (1.016%) showed a lower expression of genes such as NADH dehydrogenase subunit I (ND1), cytochrome b (CYTB) and cytochrome c oxidase subunits I (COX I), II (COX II) and III (COX III), displaying the worst performance and body protein deposition. This demonstrates the relationship existing between the expression of the evaluated genes and the performance responses. In conclusion, results indicate that broilers fed diets with higher levels of digestible lysine have increased messenger RNA expression of some genes coded in the mitochondrial electron transport chain (ND1, CYTB, COX I, COX II and COX III). It may be stated that diets with proper levels of digestible lysine, within the ‘ideal protein’ concept, promote the expression of genes, which increases the mitochondrial energy, thereby fostering body protein deposition and the performance of broilers in the starter phase.     

Characteristics of external and internal NAD(P)H dehydrogenases in Hoya carnosa mitochondria

This study aims at characterizing NAD(P)H dehydrogenases on the inside and outside of the inner membrane of mitochondria of one phosphoenolpyruvate carboxykinase??crassulacean acid metabolism plant, Hoya carnosa. In crassulacean acid metabolism plants, NADH is produced by malate decarboxylation inside and outside mitochondria. The relative importance of mitochondrial alternative NADH dehydrogenases and their association was determined in intact??and alamethicin??permeabilized mitochondria of H. carnosa to discriminate between internal and external activities. The major findings in H. carnosa mitochondria are: (i) external NADPH oxidation is totally inhibited by DPI and totally dependent on Ca²⁺, (ii) external NADH oxidation is partially inhibited by DPI and mainly dependent on Ca²⁺, (iii) total NADH oxidation measured in permeabilized mitochondria is partially inhibited by rotenone and also by DPI, (iv) total NADPH oxidation measured in permeabilized mitochondria is partially dependent on Ca²⁺ and totally inhibited by DPI. The results suggest that complex I, external NAD(P)H dehydrogenases, and internal NAD(P)H dehydrogenases are all linked to the electron transport chain. Also, the total measurable NAD(P)H dehydrogenases activity was less than the total measurable complex I activity, and both of these enzymes could donate their electrons not only to the cytochrome pathway but also to the alternative pathway. The finding indicated that the H. carnosa mitochondrial electron transport chain is operating in a classical way, partitioning to both Complex I and alternative Alt. NAD(P)H dehydrogenases.     

Investigating the receptor-independent neuroprotective mechanisms of nicotine in mitochondria

Although nicotine has been associated with a decreased risk of developing Parkinson disease, the underlying mechanisms are still unclear. By using isolated brain mitochondria, we found that nicotine inhibited N-methyl-4-phenylpyridine (MPP(+)) and calcium-induced mitochondria high amplitude swelling and cytochrome c release from intact mitochondria. Intra-mitochondria redox state was also maintained by nicotine, which could be attributed to an attenuation of mitochondria permeability transition. Further investigation revealed that nicotine did not prevent MPP(+)- or calcium-induced mitochondria membrane potential loss, but instead decreased the electron leak at the site of respiratory chain complex I. In the presence of mecamylamine hydrochloride, a nonselective nicotinic acetylcholine receptor inhibitor, nicotine significantly postponed mitochondria swelling and cytochrome c release induced by a mixture of neurotoxins (MPP(+) and 6-hydroxydopamine) in SH-SY5Y cells, suggesting that there is a receptor-independent nicotine-mediated neuroprotective effect of nicotine. These results show that interaction of nicotine with mitochondria respiratory chain together with its antioxidant effects should be considered in the neuroprotective effects of nicotine.     

Bioenergetics of mitochondria from flight muscles of aging blowflies: partial reactions of oxidation and phosphorylation.

Mitochondria from flight muscles of senescent blowflies, Phormia regina, which exhibit decreased rates of coupled, or ADP-stimulated, oxidation of α-glycerolphosphate or pyruvate show similar decreased rates of uncoupled, or carbonylcyanide-p-trifluoromethoxyphenylhydrazone-stimulated oxidation. Thus, uncoupled α-glycerolphosphate oxidation is decreased by 20% and uncoupled pyruvate oxidation by 39% in mitochondria isolated from 31- to 33-day-old blowflies as compared with mitochondria from 7- to 9-day-old flies. The finding of nearly equal decreases with age in coupled and uncoupled respiration suggests that the age-dependent defect lies within the oxidative or electron transport pathway, and not associated with phosphorylation. However, no such change with age is observed in any of the partial reactions of electron transport that were examined. These include the following partial reactions: (a) pyruvic dehydrogenase; (b) pyruvate-ferricyanide reductase: (c) α-glycerolphosphate dehydrogenase; (d) α-glycerolphosphate-ferricyanide reductase; and (e) cytochrome oxidase. In addition, no age-associated decline is observed in the content of cytochromes b, c + c₁, a, or a₃ or in the specific activity of the fully activated mitochondrial ATPase (assayed in the presence of carbonylcyanide-p-trifluoromethoxyphenylhydrazone). However, the specific activity of the masked ATPase (assayed in the absence of uncoupler) is increased 38% in mitochondria from senescent blowflies.     

Blood-to-brain influx transport of nicotine at the rat blood–brain barrier: Involvement of a pyrilamine-sensitive organic cation transport process

Nicotine is the most potent neural pharmacological alkaloid in tobacco, and the modulation of nicotine concentration in the brain is important for smoking cessation therapy. The purpose of this study was to elucidate the net flux of nicotine transport across the blood–brain barrier (BBB) and the major contributor to nicotine transport in the BBB. The in vivo brain-to-blood clearance was determined by a combination of the rat brain efflux index method and a rat brain slice uptake study, and the blood-to-brain transport of nicotine was evaluated by in vivo vascular injection in rats and a conditionally immortalized rat brain capillary endothelial cell line (TR-BBB13 cells) as an in vitro model of the rat BBB. The blood-to-brain nicotine influx clearance was obtained by integration plot analysis as 272 μL/(min g brain), and this value was twofold greater than the brain-to-blood efflux clearance (137 μL/(min g brain)). Thus, it is suggested that the net flux of nicotine transport across the BBB is dominated by blood-to-brain influx transport. In vivo blood-to-brain nicotine transport was inhibited by pyrilamine. [³H]Nicotine uptake by TR-BBB13 cells exhibited time-, temperature-, and concentration-dependence with a K_m value of 92 μM. Pyrilamine competitively inhibited nicotine uptake by TR-BBB13 cells with a K_i value of 15 μM, whereas substrates and inhibitors of organic cation transporters had little effect. These results suggest that pyrilamine-sensitive organic cation transport process(es) mediate blood-to-brain influx transport of nicotine at the BBB, and this is expected to play an important role in regulating nicotine-induced neural responses.