Effects Of Respiratory Inhibitors Research Articles

Effect of respiratory inhibitors and quinone analogues on the aerobic electron transport system of Eikenella corrodens

The effects of respiratory inhibitors, quinone analogues and artificial substrates on the membrane-bound electron transport system of the fastidious β-proteobacterium Eikenella corrodens grown under O2-limited conditions were studied. NADH respiration in isolated membrane particles were partially inhibited by rotenone, dicoumarol, quinacrine, flavone, and capsaicin. A similar response was obtained when succinate oxidation was performed in the presence of thenoyltrifluoroacetone and N,N’-dicyclohexylcarbodiimide. NADH respiration was resistant to site II inhibitors and cyanide, indicating that a percentage of the electrons transported can reach O2 without the bc1 complex. Succinate respiration was sensitive to myxothiazol, antimycin A and 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). Juglone, plumbagin and menadione had higher reactivity with NADH dehydrogenase. The membrane particles showed the highest oxidase activities with ascorbate-TCHQ (tetrachlorohydroquinone), TCHQ alone, and NADH-TMPD (N,N,N’,N’-tetramethyl-p-phenylenediamine), and minor activity levels with ascorbate-DCPIP (2,6-dichloro-phenolindophenol) and NADH-DCPIP. The substrates NADH-DCPIP, NADH-TMPD and TCHQ were electron donors to cyanide-sensitive cbb' cytochrome c oxidase. The presence of dissimilatory nitrate reductase in the aerobic respiratory system of E. corrodens ATCC 23834 was demonstrated by first time. Our results indicate that complexes I and II have resistance to their classic inhibitors, that the oxidation of NADH is stimulated by juglone, plumbagin and menadione, and that sensitivity to KCN is stimulated by the substrates TCHQ, NADH-DCPIP and NADH-TMPD.

Effect of respiratory inhibitors on mitochondrial complexes and ADP/ATP translocators in the Triticum aestivum roots

Effective functioning of the mitochondrial complexes of the oxidative phosphorylation (OXPHOS) system is necessary for ATP synthesis. The OXPHOS complexes exist both as individual forms and supercomplexes, whose formation and stability are supported by specific protein and lipid factors. In this paper, we report on the types and activities of OXPHOS complexes and supercomplexes from wheat (Triticum aestivum L.) root mitochondria analyzed by blue native polyacrylamide gel electrophoresis (BN-PAGE). The activity of OXPHOS complexes decreased when a mixture of rotenone, an inhibitor of complex I, and antimycin A, an inhibitor of complex III (R + AA) was applied to the BN-PAGE gels. By contrast, the types and activities of the OXPHOS complexes and supercomplexes did not change when they were isolated from the R + AA treated roots. However, the amount of the mitochondrial membrane-bound low molecular mass proteins in these roots markedly increased. The proteins were identified as ANT1 and ANT2 (ADP/ATP translocators) and ABA 8′-hydroxylase. We suggest that these low molecular mass proteins contribute to fine control mechanisms that stabilize mitochondrial supercomplexes and help to overcome an inhibitor-induced energy deficit by enhancing ADP/ATP transfer and ultimately improving the supply of ATP.

Chromium resistance characteristics of Cr(VI) resistance genes ChrA and ChrB in Serratia sp. S2

objectiveTo find an efficient chromium (VI) resistance system, with a highly efficient, economical, safe, and environmentally friendly chromium-removing strain, ChrA, ChrB, and ChrAB fragments of the chromium (VI) resistance gene in Serratia sp. S2 were cloned, and their prokaryotic expression vectors were constructed and transformed into E. coli BL21. The anti-chromium (VI) capacity and characteristics of engineered bacteria, role of ChrA and ChrB genes in the anti-chromium (VI) processes, and the mechanism of chromium metabolism, were explored. MethodsThe PCR technique was used to amplify ChrA, ChrB, and ChrAB genes from the Serratia sp. S2 genome. ChrA, ChrB, and ChrAB genes were connected to the prokaryotic expression vector pET-28a and transferred into E. coli BL21 for prokaryotic expression. Cr-absorption and Cr-efflux ability of the engineered strains were determined. The effects of respiratory inhibitors and oxygenated anions on Cr-efflux of ChrA and ChrB engineered strains were explored. ResultsChrA, ChrB, and ChrAB engineered strains were constructed successfully; there was no significant difference between the control strain and the ChrB engineered strain for Cr-metabolism (P > 0.05). Cr-absorption and Cr-efflux of ChrA and ChrAB engineered strains were significantly stronger than the control strain (P < 0.05). Oxyanions (sulfate and molybdate) and inhibitors (valinomycin and CN-) could significantly inhibit the Cr-efflux capacities of ChrA and ChrAB engineered strains (P < 0.05), while NADPH could significantly promote such capacities (P < 0.05). ConclusionThe Cr-transporter, encoded by ChrA gene, confer the ability to pump out intracellular Cr on ChrA and ChrAB engineered strains. The ChrB gene plays a positive regulatory role in ChrA gene regulation. The Cr-metabolism ability of the ChrAB engineered strain is stronger than the ChrA engineered strain. ChrA and ChrAB genes in the Cr-resistance system may involve a variety of mechanisms, such as sulfate ion channel and respiratory chain electron transfer.

Mitotic activity, submicroscopic organization and respiration of meristematic cells of root tips of Allium cepa L. treated with dacarbazine (DTIC)

Studies were carried out on the effect of dacarbazine (DTIC) on meristematic cells of root tips of &lt;em&gt;Allium&lt;/em&gt; cepa L. Noticeable mitodepression was observed. It depended upon time of exposure and DTIC concentration. Mitodepression was caused by an inhibition of DNA synthesis and blocking of the G&lt;sub&gt;2&lt;/sub&gt; phase. DTIC caused also disturbances of the karyokinesis and cytokinesis, similar as in case of colchicine effect. In the experimental conditions inhibition of cell respiration was noted, as also changes in the tissue ultrastructure. These changes were typical of the effect of respiratory inhibitors.

Inhibition of Oxidative Phosphorylation Induces a Rapid Death of GA-Pretreated Aleurone Cells, But Not of ABA-Pretreated Aleurone Cells

Reactive oxygen species (ROS) mediate programmed cell death in aleurone cells, which is promoted by gibberellic acid (GA) and prevented by abscisic acid (ABA). Plant mitochondria contain two distinct respiratory pathways: respiration through cytochrome c oxidase increases ROS production, whereas respiration through the alternative oxidase pathway lowers it. While studying the effects of GA and ABA on partitioning of respiration between those two pathways during the germinating process, we discovered that oxidative phosphorylation inhibitors like sodium azide and 2, 4-dinitrophenol induce rapid death of GA-pretreated aleurone cells but not of ABA-pretreated cells. Functional aerobic respiration was required for GA signaling, and 6 to 12 hours of GA signaling altered the cellular state of aleurone cells to be extremely susceptible to inhibition of oxidative phosphorylation. Anaerobic conditions were also able to mimic the effects of respiratory inhibitors in specifically inducing cell death in GA-treated cells, but cell death was provoked much more slowly. Cotreatment with various antioxidants did not prevent this process at all, suggesting that no ROS are responsible for this respiratory inhibitor-induced cell death. Our observation implicates that GA may partition all the electrons produced during mitochondrial respiration only to the cytochrome oxidase pathway, which would at least partly contribute to cellular accumulation of ROS.

Monitoring the in vivo redox state of plant mitochondria: Effect of respiratory inhibitors, abiotic stress and assessment of recovery from oxidative challenge

In animals, the impact of ROS production by mitochondria on cell physiology, death, disease and ageing is well recognised. In photosynthetic organisms such as higher plants, however, the chloroplast and peroxisomes are the major sources of ROS during normal metabolism and the importance of mitochondria in oxidative stress and redox signalling is less well established. To address this, the in vivo oxidation state of a mitochondrially-targeted redox-sensitive GFP (mt-roGFP2) was investigated in Arabidopsis leaves. Classical ROS-generating inhibitors of mitochondrial electron transport (rotenone, antimycin A and SHAM) had no effect on mt-roGFP oxidation when used singly, but combined inhibition of complex III and alternative oxidase by antimycin A and SHAM did cause significant oxidation. Inhibitors of complex IV and aconitase also caused oxidation of mt-roGFP2. This oxidation was not apparent in the cytosol whereas antimycin A+SHAM also caused oxidation of cytosolic roGFP2. Menadione had a much greater effect than the inhibitors, causing nearly complete oxidation of roGFP2 in both mitochondria and cytosol. A range of severe abiotic stress treatments (heat, salt, and heavy metal stress) led to oxidation of mt-roGFP2 while hyperosmotic stress had no effect and low temperature caused a slight but significant decrease in oxidation. Similar changes were observed for cytosolic roGFP2. Finally, the recovery of oxidation state of roGFP in mitochondria after oxidation by H2O2 treatment was dramatically slower than that of either the cytosol or chloroplast. Together, the results highlight the sensitivity of the mitochondrion to redox perturbation and suggest a potential role in sensing and signalling cellular redox challenge.

Alternative respiration of fungus Phycomyces blakesleeanus

Respiratory characteristics of germinating spores, developing mycelium and mitochondria of the fungus Phycomyces blakesleeanus were investigated by means of oxygen Clark-type electrode. The effects of respiratory inhibitors and metabolic compounds on oxygen consumption were tested. It was demonstrated that P. blakesleeanus apart of cyanide-sensitive respiration, CSR, possess alternative respiration, (cyanide-resistant respiration, CRR) which is constitutive and whose capacity decreases during development. Maximum is observed for activated spores where CRR capacity is significantly greater than CSR. After treatment with antimycin A, a third type of respiration insensitive to antimycin A and low concentration of SHAM (sufficient for inhibition of CRR), but sensitive to cyanide and high concentration of SHAM, has been expressed.

Using yeast to easily determine mitochondrial functionality with 1‐(4,5‐dimethylthiazol‐2‐yl)‐3,5‐diphenyltetrazolium bromide (MTT) assay

Mitochondrial respiration is an important topic in biochemical courses, but at the same time it is a very arduous and challenging subject for the students to comprehend. In teaching laboratories, experiments are also difficult to perform because mitochondrial isolation as well as oxygen consumption determinations require expensive equipment, rarely present in Mexican teaching laboratories. For this purpose, we adapted the 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenyltetrazolium bromide (MTT) assay, usually done with cell lines, to corroborate mitochondrial respiration in yeast. We used the reduction of the MTT to learn and distinguish the respiratory yield between different substrates as well as the effects of respiratory inhibitors. This is an economical, easily done laboratory experiment that quantifies mitochondrial functionality and provides a good idea of the respiratory process.

NADPH Oxidase System as a Superoxide-generating Cyanide-Resistant Pathway in the Respiratory Chain of Corynebacterium glutamicum.

The respiratory chain of Corynebacterium glutamicum was investigated, especially with respect to a cyanide-resistant respiratory chain bypass oxidase. The membranes of C. glutamicum had NADH, succinate, lactate, and NADPH oxidase activities, and menaquinone, and cytochromes a 598, b 562(558), and c 550 as respiratory components. The NADH, succinate, lactate, and NADPH oxidase systems, all of which were more cyanide-resistant than N,N,N',N'-tetramethyl-p-phenylene diamine oxidase activity (cytochrome aa 3 terminal oxidase), had different sensitivities to cyanide; the cyanide sensitivity of these oxidase systems increased in the order, NADPH, lactate, NADH, and succinate. Taken together with the analysis of redox kinetics in the cytochromes and the effects of respiratory inhibitors, the results suggested that there is a cyanide-resistant bypass oxidase branching at the menaquinone site, besides cyanide-sensitive cytochrome oxidase in the respiratory chain. H(+)/O measurements with resting cells suggested that the cyanide-sensitive respiratory chain has two or three coupling sites, of which one is in NADH dehydrogenase and the others between menaquinone and cytochrome oxidase, but the cyanide-resistant bypass oxidase may not have any proton coupling site. NADPH and lactate oxidase systems were more resistant to UV irradiation than other systems and the UV insensitivity was highest in the NADPH oxidase system, suggesting that a specific quinone resistant to UV or no such a quinone works in at least NADPH oxidase system while the UV-sensitive menaquinone pool does in other oxidase systems. Furthermore, superoxide was generated in well-washed membranes, most strongly in the NADPH oxidase system. Thus, it was suggested that the cyanide-resistant bypass oxidase system of C. glutamicum is related to the NADPH oxidase system, which may be involved in generation of superoxide anions and probably functions together with superoxide dismutase and catalase.

Reactive Oxygen Species Production in the Isolated Perfused Rat Liver: Effect of Respiratory Inhibitors.

Mitochondria are one of the possible sources of potentially harmful reactive oxygen species in tissue during ischemia-reperfusion. In order to delineate the capacity of the respiratory chain to produce reactive oxygen species, the effect of respiratory inhibitors on the oxygen uptake and luminol and lucigenin enhanced chemiluminescence was measured in isolated blood-free perfused rat livers. Concomitant tissue damage was monitored by measuring the release of lactate dehydrogenase and thiobarbituric acid reactive substances by the livers. Cyanide inhibited oxygen uptake reversibly while rotenone and Antimycin A effects were not reversible. Enhanced chemiluminescence was increased immediately by cyanide, suggesting increased formation of reactive oxygen species. Antimycin A produced a delayed, large increase in chemiluminescence concurrent with severe cell damage and increased lipid peroxidation; Cyanide and rotenone induced only limited cell damage. The cell damaging effect of Antimycin A was delayed by the simultaneous administration of cyanide. These results are discussed in terms of the possible role of reactive oxygen species produced during Antimycin A inhibition of the respiratory chain in cell damage.

Alternative respiratory pathways in Schwanniomyces castellii: involvement in energy production and growth.

Wild-type and cytochrome-deficient strains of Schwanniomyces castellii were cultivated aerobically on citrate, and the effects of respiratory inhibitors on respiration, ATP levels and energy charge were measured. The obtained results demonstrate that every respiratory pathway of Schwanniomyces castellii involves phosphorylation site I, and suggest that the functioning of this site allows the alternative pathways to produce ATP.

Initiation of lipid peroxidation in submitochondrial particles: Effect of respiratory inhibitors

Initiation of lipid peroxidation in the inner mitochondrial membrane was investigated using respiratory substrates and inhibitors and various iron chelates. An iron chelate was required for initiation of lipid peroxidation in the presence of either NADH or NADPH. The two nicotinamide nucleotides exhibited different activities in initiating lipid peroxidation with regard to concentration and to the effects of rotenone and rhein. Succinate and both nicotinamide nucleotides supported lipid peroxidation in the presence of thenoyl trifluoroacetone (TTFA), without a requirement for exogenously added iron. ADP stimulated lipid peroxidation in the case of NAD(P)H and TTFA, but inhibited it in the case of succinate and TTFA. Lipid peroxidation is thought to be enzymatically induced in both the NADH and the succinate dehydrogenase regions of the respiratory chain, and evidence is presented for a novel pathway of NADPH oxidation that may also be involved. Possible initiation mechanisms are discussed.

Effects of Respiratory Inhibitors on Red Light and Lumiflavin Induced Betacyanin Formation in Amaranthus caudatus L. Seedlings

Summary The effects of respiratory inhibitors were monitored in red light- and lumiflavin-induced betacyanin formation, using etiolated, three-day-old Amaranthus caudatus L. seedlings, derooted and decoated under green safelight. The responses to red light (5 min to 4 h) and to a cytokinin (50 nM 6-benzylaminopurine) were resistant to 1 mM KCN, while the promoting effect of lumiflavin (4 ¼M) was sensitive to 0.1 and 1 mM cyanide. Safelight control and betacyanin induction by 5 min far-red light were only partly reduced in 1 mM cyanide, which dramatically increased the far-red reversibility of red light action. These effects of cyanide, which were also observed in 0.01 mM NaN3, were apparently cancelled by 4 ¼M lumiflavin. In contrast to cyanide, 0.1 mM SHAM decreased the far-red reversibility of the red light-mediated response, while the same compound at 2 mM concentration severely suppressed the net inductive effect of prolonged red and, to a lesser extent, brief far-red light. The effects of respiratory inhibitors and lumiflavin on the rate of oxygen uptake in darkness and during 4 h illumination with red light were different in comparison to betacyanin photoregulation, but some similarity was observed during the postillumination period of 30 min darkness. These results suggest that a metabolic pattern involved in betacyanin photoregulation is restricted to a limited cell fraction in the seedlings. The relevance of such metabolic pattern for the mechanism of phytochrome action is discussed.

The effect of respiratory inhibitors on the fermentative ability of Pichia stipitis, Pachysolen tannophilus and Saccharomyces cerevisiae under various conditions of aerobiosis

The growth and ethanol production by the d-xylose-fermenting yeasts Pichia stipitis and Pachysolen tannophilus under various conditions of aerobiosis responded similarly to the addition of the respiratory inhibitors potassium cyanide (KCN), antimycin A (AA), sodium azide and rotenone. However, the d-glucose-fermenting yeast Saccharomyces cerevisiae differed markedly from these yeasts in response to the inhibitors. In general the growth of the d-xylose-fermenting yeasts was inhibited by the respiratory inhibitors while ethanol production was either stimulated (especially when oxygen was available) or unaffected or inhibited by rotenone or AA or KCN and sodium azide, respectively. However, by exception KCN and AA stimulated ethanol production under aerobic conditions by Pichia stipitis and Pachysolen tannophilus respectively. Stimulatory or inhibitory effects by respiratory inhibitors were less marked in S. cerevisiae. These data suggest that unimpaired mitochondrial function is necessary for growth on d-xylose and optimal d-xylose fermentation. A requirement for membrane generated energy during d-xylose utilisation is indicated by 2,4-dinitrophenol inhibition of growth and fermentation.

RELATION BETWEEN RESPONSIVENESS TO LIGHT AND RESPIRATORY ACTIVITY IN SEEDS OF OLDENLANDIA CORYMBOSA (TROPICAL RUBIACEAE)

ABSTRACT The time course of the changes in responsiveness to light and respiratory activity of Oldenlandia corymbosa seeds was followed during their imbibition in darkness at 20°C and 35°C. The effect of respiratory inhibitors (KCN and NaN3) on responsiveness to light (4 hours of white light exposure) was also studied. At 35°C, responsiveness to light and respiratory activity increased over a period of about one day, and then decreased, the decrease in respiratory activity preceding that of responsiveness to light. At 20°C, responsiveness to light increased more slowly than at 35°C, but then remained high. The respiratory activity exhibited a similar time course. Transfer of seeds from 35°C to 20°C, or inversely, showed that the effect of both temperatures was perfectly reversible. The inhibition of respiration (by KCN or NaN3) produced a decrease in the responsiveness to light. The ascion was also completely reversible. There exists therefore a close relationship between the seed responsiveness to light ...

Peroxidase Activity in Relation to Suberization and Respiration in White Spruce (Picea glauca [Moench] Voss) Seedling Roots

Peroxidase (EC 1.11.1.7) activity is associated with suberization during endodermal development and metacutization in roots of white spruce (Picea glauca [Moench] Voss) seedlings. Histochemical analysis indicates a relationship between suberization and peroxidase activity, but peroxidase is ubiquitous. Increased peroxidase activity results from the induction of four anodic peroxidase isozymes in addition to quantitative increases in two anodic peroxidase isozymes. Four of these polymerized eugenol. Cold temperatures induce formation of two anodic isozymes and result in suberization. The increased peroxidase activity associated with suberization is correlated to residual respiration. In an attempt to elucidate this relationship, the effect of respiratory inhibitors on respiration and peroxidase activity are compared.

Phenazine methosulfate stimulation of ouabain-sensitive Rb+ uptake by HeLa cells: effects of respiratory inhibitors, anaerobiosis, and ascorbate.

phenazine methosulfate (PMS) stimulates ouabain-sensitive Rb+ uptake by HeLa cells. This stimulation cannot be attributed to the effect of the dye on the intracellular Na+ or ATP content. Respiratory inhibitors, such as 5 mM NaCN and 5 microM rotenone, and anaerobic conditions enhance the stimulation of Rb+ uptake by PMS. Cellular respiration is stimulated, but lactate production is reduced in the presence of PMS, irrespective of the presence of respiratory inhibitors. Cellular NADH is oxidized markedly on addition of PMS plus inhibitors, but it is not affected by addition of the inhibitors only. In the presence of a high concentration of PMS, PMS-stimulated ouabain-sensitive Rb+ uptake is inhibited by addition of ascorbate. From these results it is concluded that Na+K-pump activity is closely related to the cellular redox state.

Effects of Respiratory Inhibitors on Respiration, ATP Contents, and the Circadian Conidiation Rhythm of Neurospora crassa

Effects of respiratory inhibitors on the circadian clock, respiratory activity, and ATP content were examined in Neurospora crassa. All inhibitors, potassium cyanide, sodium azide, antimycin A, and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), shifted the phase of the conidiation rhythm. All the phase response curves were similar and resembled that for cycloheximide, but were different from the phase response curve for light. Phase shifting by azide and CCCP was proportional to the lowering of respiratory activity and ATP content, but such a correlation was not observed for cyanide and antimycin A. In particular, cyanide at a concentration of 0.5 millimolar completely depleted ATP of the cultures but did not significantly shift their phase. Their results suggest that large shifts caused by these inhibitors are not due to a decrease in energy from respiratory activity.

The effect of respiratory inhibitors during growth and development of Dictyostelium discoideum

Two major pathways of electron transport to oxygen were identified in intact cells of Dictyostelium discoideum, a cyanide-sensitive pathway and a cyanide-insensitive, salicylhydroxamic-acid-sensitive pathway. The extent to which each pathway contributed to the total respiratory activity was shown to change during exponential growth and throughout development. During exponential growth both pathways appear to be utilized to varying degrees dependent on culture age, during late exponential growth the activity of the salicylhydroxamic-acid-sensitive pathway would seem to be almost totally lost. During development the cyanide-sensitive pathway appears to be dominant up to the aggregation stage, but both pathways are active in pseudoplasmodial cells. It is also suggested that the presence of iron in the growth medium may be essential directly or indirectly, for the maintained activity of the salicylhydroxamic-acid-sensitive pathway late in exponential growth.

Effects of respiratory inhibitors and ionophore A23187 on frog gustatory nerve responses

1. 1. An increase in Ca concentration in a frog taste cell by application of respiratory inhibitors and ionophore A23187 to Ringer solution perfusing the lingual artery led to a large suppression of the taste nerve responses to quinine, ethanol and acids. 2. 2. The responses to CaCl 2, l-threonine, d-galactose and distilled water were unchanged or increased.