Ferricyanide Reductase Research Articles

The complexity of the inhibitory action of dicoumarol on the mixed function oxidase system of rat liver microsomes

The inhibitory effect of dicoumarol on the mixed function oxidase system of liver microsomes from phenobarbital treated rats were studied. Lineweaver-Burk plots of p-nitroanisole O-demethylation were non-linear in the presence of dicoumarol but linear in its absence. At high concentrations of p-nitroanisole the inhibition was partially overcome indicating a competition between the substrate and dicoumarol for the enzyme. Furthermore, it was found that dicoumarol promotes a time and concentration dependent conversion of cytochrome P-450 to P-420 and that it inhibits cytochrome P-450 reduction. While these fenomena seem directly related to the inhibitory action of dicoumarol on the overall p-nitroanisole demethylase reaction, an inhibitory effect of dicoumarol on NADH : ferricyanide reductase, on the contrary, was shown not to be related to the inhibition of the demethylation process.

Studies on reduced pyridine nucleotide dehydrogenase in bovine erythrocytes II. Electron acceptor specificity of two types of reduced pyridine nucleotide dehydrogenase in bovine erythrocytes

Two types of reduced pyridine nucleotide dehydrogenase have been characterized in erythrocytes with respect to electron acceptors and donors. Both enzymes have reducing activity towards dyes such as 2,6-dichlorophenolindophenol (DCIP) and methylene blue. Cytochrome b 5 and ferricyanide reductase activity are characteristic to only one of the dehydrogenases. The Reduced pyridine nucleotide specificity of the two enzymes was different. The NADH/NADPH ratio for the former enzyme was 2.9 at pH 7.5; the latter enzyme was specific for NADH only. Methemoglobin prepared by various oxidizing methods was directly reduced by the enzyme plus NADH only. Methylene blue and DCIP effected methemoglobin reduction but the effect of the dyes was different for each enzyme.

The purification and properties of the respiratory-chain reduced nicotinamide--adenine dinucleotide dehydrogenase of Torulopsis utilis.

1. An NADH-ferricyanide reductase activity has been isolated from the respiratory chain of Torulopsis utilis by using detergents. The isolated enzyme contains non-haem iron, acid-labile sulphide and FMN in the molar proportions 27.5:28.4:1. The preparation is free of FAD and largely free of cytochrome. 2. The enzyme catalyses ferricyanide reduction by NADPH at about 1% of the rate with NADH, and reacts poorly with acceptors other than ferricyanide. The rates of reduction of some acceptors are, as percentages of the rate with ferricyanide: menadione, 0.35%; lipoate, 0.01%; cytochrome c, 0.065%; dichlorophenolindophenol, 0.35%; ubiquinone-1, 0.08%. 3. Several properties of submitochondrial particles of T. utilis (non-haem iron, acid-labile sulphide, FMN and an NADH-reducible electron-paramagnetic-resonance signal) were found to co-purify with the NADH-ferricyanide reductase activity. Thus about 70% of the FMN and, within the limits of accuracy of the experiments, 100% of the non-haem iron and acid-labile sulphide of submitochondrial particles derived from T. utilis cells grown under conditions of glycerol limitation (but relatively low iron availability) can be attributed to the NADH-ferricyanide reductase. 4. It was also shown that the component of submitochondrial particles specifically bleached at 460nm by NADH [species 1 of Ragan & Garland (1971)] co-purifies with the NADH-ferricyanide reductase. 5. This successful purification of an NADH dehydrogenase from T. utilis forms a starting point for investigating the molecular properties of phenotypically modified mitochondrial NADH oxidation pathways that lack energy conservation between NADH and the cytochromes.

Degradation and restoration of mitochondria upon deaeration and subsequent aeration of aerobically grown Saccharomyces Cerevisiae cells

Changes in the mitochondria of aerobically grown Saccharomyces cerevisiae cells upon deaeration and subsequent aeration of the medium were studied. 1. It is shown that removal of oxygen at the end of the exponential phase of growth (after completion of mitochondria formation) causes a decrease in activity of the respiratory enzymes. The activity of the complete respiratory system decreases much more rapidly than the activities of its fragments (NADH: ferricyanide reductase, succinate:ferricyanide reductase, NADH:cytochrome c reductase, succinate:cytochrome c reductase and cytochrome oxidase). The activities are restored to their initial level upon aeration of the cell suspension. The addition of Tween-80 and ergosterol to the medium prior to deaeration does not prevent inactivation of the respiratory system. All the changes in mitochondria described occurred under conditions where cell division was insignificant. 2. Deaeration of the medium decreases the content of cytochromes b and aa 3 in the mitochondrial fraction, cytochrome aa 3 “disappearing” more quickly. The concentration of cytochromes in this fraction increases upon subsequent aeration of the cells. The total cytochromal content of the cells remains practically unchanged under the same conditions. 3. According to electron microscopic data, anaerobiosis causes a certain disorganization of mitochondrial cristal membranes. The mitochondrial structures are recovered upon aeration of the yeast cell suspension. It may be reasoned that inactivation and reactivation of the respiratory system are associated with reversible changes in mitochondrial membrane structure. 4. The effect of protein synthesis inhibitors on the restoration of mitochondria was investigated. It is shown that chloramphenicol does not suppress this process. In the presence of cycloheximide, oxygen induces reactivation of the respiratory system and simultaneously the appearance of particles resembling mitochondria. However, these particles gradually undergo morphological changes and the respiratory activity of the mitochondrial fraction decreases. Cycloheximide added to yeast cells that had not been deaerated, did not affect their mitochondria. 5. The results described suggest that the functions of oxygen in the formation of mitochondria are not restricted to the induction of mitochondrial protein synthesis and to the participation in the synthesis of certain non protein membrane components. Evidently, oxygen has a direct effect on the assembly of the respiratory system and mitochondrial membranes as a whole.

An atypical substrate-inhibitor-rate relationbship for the action of antimycin A on the succinate-ferricyanide reductase activity of beef heart electron transfer particles.

The degree of inhibition of the succinate–ferricyanide reductase activity of beef heart electron transfer particles by antimycin A is negligible at low succinate concentrations (< 1 mM) but increases to about 40% at saturating concentrations of succinate. This behavior is explained on the basis of a random order mechanism for the primary dehydrogenase with alternative routes from the monosubstrate complexes, only one of which is sensitive to antimycin.

Electron transport activities in rat liver endoplasmic reticulum membrane fragments prepared by chromatography on Sepharose 2B

A rapid and simple method based on Sepharose 2B column chromatography of the postmitochondrial supernatant is presented for isolation of endoplasmic reticulum membrane fragments from rat liver. Microsomal electron transport activities (NADPH—cytochrome c reductase, NADH — cytochrome c reductase, NADH—ferricyanide reductase and NADPH oxidase) in preparations so obtained are fully comparable with the activities of preparations obtained by ultracentrifugation.

Mixed-function oxidation in tumors.

Liver microsomes of the rat contain a group of hydroxylating enzymes which are coupled to a greater or lesser degree to the electron flow system. In our studies, enzymes believed to be directly associated with the electron flow chain of NADPH, ferricyanide reduction, cytochrome c, cytochrome P-450 and substrate hydroxylation have been observed in livers obtained from normal, tumor-bearing and whole body irradiated rats as well as in Morris hepatoma 7777 and dimethyl-amino-biphenyl induced breast tumors.A significant difference appeared to exist in the activity of NADPH oxidase, NADP-ferricyanide reductase and benzopyrene hydroxylase when normal liver was compared with the liver obtained from a breast-tumor-bearing animal. Both cytochrome P-450 and cytochrome b(5) were decreased in the tumor-bearing animal.Tissue distribution of benzopyrene hydroxylase in normal, lactating and tumor-bearing Wistar rats has been studied.With the exception of NADPH oxidase, the activities of NADP-cytochrome c reductase, NADPH-ferricyanide reductase, benzopyrene hydroxylase and P-450 were markedly different in liver from Morris hepatoma 7777-bearing Buffalo rat when this was compared with homologous tissue obtained from normal Buffalo rat.Whole-body irradiated animals showed increased P-450 and NADPH oxidase activity in liver as a function of irradiation and there further appeared to be a correlation with decreased ferricyanide reductase activity.

Isolation of the Cadmium Derivative of Lipoyl Dehydrogenase

Abstract Reduced lipoamide dehydrogenase reacts with cadmium ion to form a moderately stable compound characterized by 1 g atom of cadmium bound per mole of flavin adenine dinucleotide, a modified spectrum in the visible region, and altered reactivity with electron acceptor substrates. The 450-mµ band is shifted to the blue and the 370-mµ shoulder is lost. On storage, the spectrum of the cadmium-lipoyl dehydrogenase complex tends to return to that of the unmodified enzyme. Alkylated lipoyl dehydrogenase, inactive in the DPNH-lipoamide dehydrogenase reaction, forms the corresponding cadmium compound. The latter appears more stable than the cadmium compound of the unalkylated enzyme. The activity profile of the cadmium compound is characterized by increased rates of 2,6-dichloroindophenol and quinone reductase, little change in ferricyanide reductase, decreased pyridine nucleotide transhydrogenase, and highly inhibited DPNH-lipoamide dehydrogenase activity.

Quinone interaction with the respiratory chain-linked NADH dehydrogenase of beef heart mitochondria I. Juglone reductase activity

1. Substituted benzoquinones and napthoquinones can function as electron acceptors for the NADH dehydrogenase segment of the mitochondrial electron transport system. Anthraquinones and several hydroxylated quinones are not reduced by the enzyme complex. 2. Piericidin A treatment at concentrations known to inhibit electron transport causes varying degrees of inhibition of quinone reductase activities. The pattern of piericidin A inhibition suggests that certain quinones are reduced at sites either before or after the piericidin A inhibition site. Reduction of quinones such as 5-hydroxy-1,4-napthoquinone (juglone) and 1,2-napthoquinone is inhibited only slightly. Reduction of ubiquinone 1 and 2 and 2,3,5,6-tetramethyl-1,4-benzoquinone (duroquinone) is almost completely inhibited following piericidin A treatment. 3. Comparison of juglone reductase activity with ferricyanide reductase activity suggests that these acceptors are reduced at nonequivalent sites in the NADH dehydrogenase. Juglone reductase activity is stimulated following mercurial treatment while ferricyanide reductase activity is slightly inhibited. Preincubation of NADH dehydrogenase with NADH followed by mercurial treatment causes almost complete inhibition of ferricyanide reductase activity. Activation of juglone reductase activity still occurs under these conditions. The activation is specific for preparations of NADH dehydrogenase, for mercurials, and for napthoquinone reductase activity.

Regulation of oxidase enzyme systems in trypanosomes

1. 1. QO 2, succinoxidase and glycerophosphate oxidase (GPO) levels (0·533, 0·367 and 0·334 μmoles O 2 hr −1 mg protein −1) were determined for Trypanosoma conorhini. 2. 2. Kinetoplastic and dyskinetoplastic T. equiperdum had higher QO 2 (2·51 and 1·87) and GPO (2·38 and 1·15) levels but lacked measurable succinoxidase. 3. 3. Reduced O 2 availability during growth of T. conorhini gave reduced succinoxidase and increased GPO activities. 4. 4. Acriflavin and chloramphenicol added at initiation of cultures of T. conorhini gave decreased growth and QO 2 while only acriflavin decreased succinate ferricyanide reductase activity. 5. 5. Acriflavin added to exponentially growing T. conorhini reduced only succinate ferricyanide reductase activity and increased cyanide-insensitive QO 2 and GPO levels.

Inhibitors and Activators of the Mitochondrial Reduced Diphosphopyridine Nucleotide Dehydrogenase

Abstract The inhibitor response properties of the mitochondrial DPNH dehydrogenase have been studied with both the particle-bound and the soluble forms of the enzyme. Electron transfer from the particle-bound DPNH dehydrogenase to the respiratory chain is inhibited by barbiturates, Demerol, rotenone, and piericidin A, whereas the activities of the soluble enzyme are essentially unaffected by these inhibitors. In contrast, the ferricyanide reductase, as well as other diaphorase activities, of the soluble DPNH dehydrogenase is inhibited by mercurials, whereas the bound dehydrogenase appears to be protected against such mercurial attack. o-Phenanthroline interacts with the soluble dehydrogenase, inhibits its activities, and causes the release of labile sulfide from the enzyme. However, other bidentate iron chelators such as bathophenanthroline sulfonate and Tiron do not inhibit the enzyme but interact with it and protect it against inhibition by o-phenanthroline. These studies have indicated that in DPNH dehydrogenase the complex among iron, labile sulfide groups, and cysteine sulfur residues must have a tetrahedral structure. Adenosine phosphates inhibit DPNH dehydrogenase in the same manner as does DPN, but adenosine, nicotinamide mononucleotide, and NMNH are not inhibitory. Guanidinium compounds activate the soluble DPNH dehydrogenase, but inhibit the coenzyme Q reductase activity of the particulate DPNH-coenzyme Q reductase (Complex I). The activating effect of guanidinium compounds on the soluble dehydrogenase is associated not only with an increase in the value of Vmax, but also with a substantial decrease in the value of Kmdpnh (3-fold at 50 mm guanidine).

The inhibition of mammalian mitochondrial NADH oxidation by chloramphenicol and its isomers and analogues.

Chloramphenicol and its isomers and analogues have been found to inhibit the oxidation of NADH, but not that of succinate, by beef heart mitochondria. They must therefore inhibit the NADH dehydrogenase segment of the respiratory chain. Chloramphenicol gave 50% inhibition at a concentration of 1 mM. The methylthio analogue of chloramphenicol inhibited NADH – coenzyme Q6 reductase but not NADH–ferricyanide reductase. Spectrophotometric observations suggest that these inhibitors act between NADH and flavin in coupled rat liver mitochondria and between flavin and cytochrome b in uncoupled beef heart mitochondria.

Aerobic glycolysis, oxygen uptake, and enzyme activity of the electron transport system in neoplastic and non-neoplastic ovarian tissue grafted into the spleen of C57BL mice

Summary The lactic acid production, oxygen uptake, and activities of 5 enzymes of the electron transport system have been studied in normal ovaries, 60-day-old intrasplenic grafted ovarian tissue, and primary 12-month-old ovarian tumors developed from grafts in the spleen of C57BL mice. The 60-day-old grafted ovarian tissue, although not greatly different from normal ovaries, seems to possess quite a distinct biochemical pattern. Decreased activity in the following parameters were found: oxygen uptake, aerobic lactic acid production, reduced diphosphopyridine nucleotide oxidase, reduced diphosphopyridine nucleotide-cytochrome c reductase, reduced diphosphopyridine nucleotide ferricyanide reductase, succinate-cytochrome c reductase, and cytochrome oxidase. In the primary tumors a lower oxygen uptake coincident with a decreased level of succinate-cytochrome c reductase and cytochrome oxidase, compared with normal ovaries, was observed. Glucose utilization was displayed only by the primary tumors. The data obtained suggest that in the 60-day-old grafts a respiratory damage is operating, possibly as a first step toward the neoplastic change of the ovarian cell. Based on these findings it can be stated that the Warburg hypothesis on the origin of the cancer cell seems to be supported for the ovarian tissue in the preneoplastic stage.

Studies on the respiratory chain-linked reduced nicotinamide-adenine dinucleotide dehydrogenase I. Effect of diethyl ether on particulate NADH dehydrogenase

1. The effect of diethyl ether on particulate NADH dehydrogenase of heart-muscle preparation was studied. The solvent caused a rapid and complete inhibition of NADH oxidation by oxygen, cytochrome c, Q-1 and Q-6 both when used as an extractant and when dissolved in the reaction medium at low concentrations. The ether treatment did not affect NADH-ferricyanide reductase and succinate oxidase (in the presence of added cytochrome c). Both menadione and 2,6-dichlorophenol-indophenol (DCIP) reductase were inhibited by about 35%. 2. The effect of ether on enzymic activities was similar to the effect of short-term digestion of the preparation with small amounts of phospholipase. 3. High concentrations of rotenone, o-phenanthroline, N-ethylmaleimide and of p-chloromercuribenzoate (PCMB) did not affect all ferricyanide, menadione and DCIP activities of the ether-extracted preparation. 4. Treatment of the preparation with ether protected NADH dehydrogenase against inactivation by preincubation with NADH. Sensitivity of Type III -SH groups towards mersalyl was also reduced. 5. Specific activities of soluble preparation obtained by the method of Mahler were 2–3 times higher when ether-pretreated preparation was used as a source of the enzyme. 6. The results indicate that ether and phospholipase displayed similar effects on the electron transport chain in respect to both the site and the mechanism of action. Both reagents did not affect ferricyanide reductase activity, which is characteristic of NADH dehydrogenase. Ether-treated preparations were modified in such a way that the extraction of soluble NADH dehydrogenase by chemical agents was facilitated.

The action of amytal on frog gastric mucosa

The frog gastric mucosa has been shown to be sensitive to amytal. At 2 mM acid secretion was completely inhibited with a rise of resistance, fall in short-circuit current and no significant change in potential difference. Simultaneously there was 75% inhibition of O 2 consumption and 50% depression of ATP levels. Dual-beam spectrophotometric studies of intact mucosa with amytal showed a crossover point between NAD + and FAD. The microsomal NADH oxidase ferricyanide reductase has also been shown to be amytal sensitive. Cl − transport was relatively insensitive to amytal, suggesting a qualitative distinction between the mechanisms underlying the transport of H + and Cl − in the mucosa. This was further brought out by the effects of anoxia in which H + transport was inhibited at 5 min but Cl − transport at minimally 20 min following the onset of anoxia.

Studies on the Triphosphopyridine Nucleotide-Cytochrome f Reductase of Chloroplasts

The reduced triphosphopyridine nucleotide-cytochrome f reductase of chloroplasts is shown to be identical with TPNH diaphorase, transhydrogenase, and ferredoxin-TPN reductase. The crystalline enzyme, aflavin adenine dinucleotide-flavoprotein, has a molecular weight of 40,000 to 45,000. Turnover numbers of 4,600 and 18,000 are calculated for TPNH-cytochrome f reductase and ferricyanide reductase activities, respectively. The Km values for TPNH-ferricytochrome f and ferricyanide are reported. The mechanism of ferricytochrome f and of ferricyanide reactions seem to differ on the basis of reaction kinetics. The former, but not the latter, seem to involve the formation of a ternary complex of enzyme with both substrates. The enzyme is reversibly inactivated by preincubation with TPNH; TPN prevents and reverses inactivation. Relatively low concentrations of urea convert the reversible inactivation by TPNH into an irreversible process. The function of FAD and urea-sensitive bonds in the preservation of the native structure of the enzyme is discussed on the basis of these findings.

RESPIRATORY PATHWAYS IN THE MYCOPLASMA. I. LACTATE OXIDATION BY MYCOPLASMA GALLISEPTICUM.

Smith, S. L. (Cornell University, Ithaca, N.Y.), P. J. Van Demark, and J. Fabricant. Respiratory pathways in the Mycoplasma. I. Lactate oxidation by Mycoplasma gallisepticum. J. Bacteriol. 86:893-897. 1963.-Resting cells of Mycoplasma gallisepticum 293 required the addition of nicotinamide adenine dinucleotide, thiamine pyrophosphate, and flavine mononucleotide for the maximal rate of sodium lactate oxidation. Inhibitor studies, as well as spectrophotometric and chemical assays, indicate that the pathway of electron transport to oxygen during lactate oxidation does not involve heme catalysts, and is mediated by flavin-linked enzyme systems. The presence of reduced nicotinamide adenine dinucleotide-specific lactic dehydrogenase, menadione reductase, ferricyanide reductase, and reduced nicotinamide adenine dinucleotide oxidase activities was detected in cell-free extracts. No cytochrome c reductase or reduced nicotinamide adenine dinucleotide peroxidase activity was detected in these extracts.

Quinones in electron transport. II. Influence of accessory lipid factors on the activity of coenzyme Q analogs

The restoration of succinoxidase and succinic ferricyanide reductase in acetone-extracted electron-transport particles from beef heart mitochondria has been studied. In the succinoxidase system, coenzyme Q 10 and analogs with long isoprenoid chains require addition of accessory lipid material in order to be fully effective, whereas short-chain analogs are more effective without accessory lipids. The action of the accessory lipids is related to a positioning effect on the Q analogs, and it appears that no additional electron carrier function other than coenzyme Q is involved.