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.     

Molecular biology of plasma membrane redox enzymes: A survey of current knowledge

Summary Despite a large body of evidence for enzymatic activities and physiological functions of plasma membrane redox function, few of these enzymes have been characterized in terms of molecular biology. Examples for these with at least some molecular data up to complete sequences, membrane topology and binding sites for substrates and coenzymes or prosthetic groups are NADH-ferricyanide reductase of Ehrlich ascites membranes, NADH-coenzyme Q reductase of liver, NADH oxidase ectoenzyme of liver and HeLa (and possibly other) cells, protein disulfide isomerase which is widespread, and relatives thereof, as well as cytochromes P-450 andb ₅₅₈, NADPH oxidase of fat and thyroid cells and fat cell amine oxidase. Ferricyanide reductase and coenzyme O reductase may be identical, but NADH oxidase ectoenzyme is distinct and possibly functions also as a disulfide and a copper reductase. On the other hand, the plasma-membrane-located protein disulfide isomerase (PDI), despite its similar enzymatic activity, is completely different from the ectooxidase. The latter is shed from the membrane into the surrounding medium by proteolysis, whereas PDI is not an integral membrane protein and is secreted intact. Another disulfide reductase has been demonstrated in THP-1 cells, which again is totally different from the former two. It turns out that enzymatic activities are insufficient to describe redox enzymes. Special forms of cytochrome P-450 can be induced to expression at the cell membrane of liver, where they are transported by the cytoskeleton-associated secretory pathway. Why some isoforms are expressed at the surface and some are not is not yet clear. Cytochromeb ₅₅₈, the flavocytochrome of neutrophils, is described in other cells too, but there are different isoforms, which are genetically distinct. A relative has also been identified in duodenal cells, where it functions as a ferric reductase involved in iron absorption. NADPH oxidase of fat cells has very similar properties, but the identity is unproved, whereas thyroid oxidase is a non-heme protein which is calcium-sensitive and does not need assembly of subunits for activation. Finally, fat cell membranes also possess a quinone-containing amine-oxidase which may be involved in signaling of glucose-transport regulation, as it is also found in GLUT4-containing vesicles. However, the physiological connection has yet to be demonstrated.     

Intermolecular electron transfer in two-iron superoxide reductase: a putative role for the desulforedoxin center as an electron donor to the iron active site

Superoxide reductase (SOR) is a superoxide detoxification system present in some microorganisms. Its active site consists of an unusual mononuclear iron center with an FeN4S1 coordination which catalyzes the one-electron reduction of superoxide to form hydrogen peroxide. Different classes of SORs have been described depending on the presence of an additional rubredoxin-like, desulforedoxin iron center, whose function has remained unknown until now. In this work, we investigated the mechanism of the reduction of the SOR iron active site using the NADPH:flavodoxin oxidoreductase from Escherichia coli, which was previously shown to efficiently transfer electrons to the Desulfoarculus baarsii SOR. When present, the additional rubredoxin-like iron center could function as an electronic relay between cellular reductases and the iron active site for superoxide reduction. This electron transfer was mainly intermolecular, between the rubredoxin-like iron center of one SOR and the iron active site of another SOR. These data provide the first experimental evidence for a possible role of the rubredoxin-like iron center in the superoxide detoxifying activity of SOR.     

Dopamine Promotes the Survival of Embryonic Striatal Cells: Involvement of Superoxide and Endogenous NADPH Oxidase

The dopaminergic system appears early in mammalian brain development, and a neurodevelopmental role for dopamine (DA) has been suggested. In the present study, we found that DA markedly promoted the survival of embryonic striatal cells in cultures. The failure of DA receptor antagonists to block this survival-promoting effect and the capability of S-apomorphine, which is devoid of DA receptor agonist activity but possesses antioxidative activity as R-apomorphine and DA, to completely mimic this effect suggested that DA receptor activation was not required in the survival-promoting effect elicited by DA, and its antioxidative activity might be involved. Moreover, it was found that mRNA of NADPH oxidase was expressed in the embryonic striatum. Furthermore, DPI or apocynin, NADPH oxidase inhibitors, promoted the survival of embryonic striatal cells. Addition of either DA or DPI into striatal cell cultures decreased the superoxide level. These results indicate that the mechanisms underlying the neuroprotective effects of DA were likely associated with its antioxidative activity. NADPH oxidase might contribute, at least in part, to ROS generation.     

Activity of neutrophil NADPH oxidase in iron-deficient anemia

This study was designed to measure the effects of iron supplementation on respiratory burst in iron-deficient anemia. The performance of neutrophils was evaluated by measuring the activity of NADPH oxidase in 18 patients with iron-deficient anemia before and after body iron stores are saturated. The activity of NADPH oxidase was significantly lower in pretreatment patients relative to controls (p<0.05). The activity increased after iron supplementation to levels that had no significant differences relative to controls.     

Fluorescence spectroscopic detection of mitochondrial flavoprotein redox oscillations and transient reduction of the NADPH oxidase-associated flavoprotein in leukocytes

Steady-state and time-resolved fluorescence spectroscopy and fluorescence microscopy of leukocyte flavoproteins have been performed. Both living human peripheral blood monocytes and neutrophils have been utilized as experimental models, as the former relies much more heavily on mitochondrial metabolism for energy production than the latter. We confirm previous studies indicating that cellular flavoproteins absorb at 460 nm and emit at 530 nm, very similar to that of the FAD moiety. Furthermore, the emission properties of intracellular flavoproteins were altered by the metabolic inhibitors rotenone, antimycin A, azide, cyanide, DNP (2,4-dinitrophenol), and FCCP [carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone]. Kinetic studies revealed flavoprotein emission oscillations in both monocytes and neutrophils. The flavoprotein intensity oscillations correlated with the physiological status of the cell and the nature of membrane receptor ligation. Microscopy revealed the presence of flavoprotein fluorescence in association with the plasma membrane, intracellular granules and distributed throughout the cytoplasm, presumably within mitochondria. Metabolic inhibitors such as cyanide suggest that the plasma membrane and granular components are cyanide-insensitive and therefore are likely associated with the flavoprotein component of the NADPH oxidase, which is located in these two compartments. This interpretation was found to be consistent with structural localization of the NADPH oxidase using an antibody molecule specific for this protein. Using peripheral blood neutrophils, which display less active mitochondria, and time-resolved emission spectroscopy, we show that the NADPH oxidase-associated flavoprotein undergoes a periodic transient reduction of about 54±2 ms in living cells. This finding is consistent with prior studies indicating that propagating substrate (NADPH) waves periodically promote electron transport across the NADPH oxidase.     

Reconstituted high-density lipoprotein suppresses leukocyte NADPH oxidase activation by disrupting lipid rafts

Reconstituted discoidal high-density lipoprotein (rHDL) has potent vascular protective actions. Native HDL suppresses cellular generation of reactive oxygen species, whereas this antioxidant effect of rHDL is less clear. This study examined the effects of rHDL on NADPH oxidase, a major source of cellular superoxide generation, in both leukocytes and human umbilical vein endothelial cells. Superoxide was measured with lucigenin-enhanced chemiluminescence. Expression of NADPH oxidase sub-units was determined by real-time PCR. Pre-treatment of HL-60 cells with rHDL (10 and 25 µM) for 1 h significantly reduced phorbol 12-myristate 13-acetate-stimulated superoxide production. Treatment with rHDL for up to 24 h did not change the mRNA expression of NADPH oxidase sub-units. In HL-60 cells, depletion of cholesterol from the plasma membrane by methyl-β-cyclodextrin mimicked the effect of rHDL, whereas cholesterol repletion blunted the effects of rHDL. Treatment with rHDL induced disruption of the lipid raft structures and blunted PMA-induced redistribution of p47phox into lipid rafts. In contrast, treatment of endothelial cells with rHDL for up to 18 h had no effect on either basal or tumour necrosis factor-α-stimulated NADPH oxidase activity, but markedly suppressed the cytokine-induced expression of proinflammatory adhesion molecules. The results suggest that rHDL inhibits NADPH oxidase activation in leukocytes, probably by interrupting the assembly of NADPH oxidase sub-units at the lipid rafts. This effect may contribute to the vascular protective actions of rHDL against inflammation-mediated oxidative damage.     

Redox enzymes in the plant plasma membrane and their possible roles

Purified plasma membrane (PM) vesicles from higher plants contain redox proteins with low‐molecular‐mass prosthetic groups such as flavins (both FMN and FAD), hemes, metals (Cu, Fe and Mn), thiol groups and possibly naphthoquinone (vitamin K₁), all of which are likely to participate in redox processes. A few enzymes have already been identified: Monodehydroascorbate reductase (EC 1.6.5.4) is firmly bound to the cytosolic surface of the PM where it might be involved in keeping both cytosolic and, together with a b‐type cytochrome, apoplastic ascorbate reduced. A malate dehydrogenase (EC 1.1.1.37) is localized on the inner side of the PM. Several NAD(P)H‐quinone oxidoreductases have been purified from the cytocolic surface of the PM, but their function is still unknown. Different forms of nitrate reductase (EC 1.6.6.1–3) are found attached to, as well as anchored in, the PM where they may act as a nitrate sensor and/or contribute to blue‐light perception, although both functions are speculative. Ferric‐chelate‐reducing enzymes (EC 1.6.99.13) are localized and partially characterized on the inner surface of the PM but they may participate only in the reduction of ferric‐chelates in the cytosol. Very recently a ferric‐chelate‐reducing enzyme containing binding sites for FAD, NADPH and hemes has been identified and suggested to be a trans‐PM protein. This enzyme is involved in the reduction of apoplastic iron prior to uptake of Fe²⁺ and is induced by iron deficiency. The presence of an NADPH oxidase, similar to the so‐called respiratory burst oxidase in mammals, is still an open question. An auxin‐stimulated and cyanide‐insensitive NADH oxidase (possibly a protein disulphide reductase) has been characterized but its identity is still awaiting independent confirmation. Finally, the only trans‐PM redox protein which has been partially purified from plant PM so far is a high‐potential and ascorbate‐reducible b‐type cytochrome. In co‐operation with vitamin K₁ and an NAD(P)H‐quinone oxidoreductase, it may participate in trans‐PM electron transport.     

Temperature profile of oxygen activation and defense against oxygen toxicity in a psychrophilic member of the Bacteroidaceae

Abstract The temperature profiles have been determined for O₂ reduction by activating substrates for whole cells and cell extracts of the psychrophilic, obligately anaerobic bacterium, strain B6, belonging to the Bacteroidaceae. The profiles were similar whether the cells were grown at 15 or 1°C, and also for cells harvested in the exponential or stationary phase. The H₂O producing pyruvate oxidase displayed in cell-free extracts a considerably higher activity than the H₂O₂ producing NADH and NADPH oxidases at all temperatures in the range 30–1°C, and characteristically makes up a larger proportion of the total O₂ reduction capacity the lower the temperature. It thus seems that the O₂ scavenging property of the pyruvate oxidase, postulated to be utilized in a defense mechanism against the detrimental effects of the H₂O₂ producing pyridine nucleotide oxidases, is particularly well adapted to function at the low temperatures of the Barents Sea, from which this obligately anaerobic organism originates.     

Paraquat and iron-dependent lipid peroxidation

The aim of this work was to study the effect of paraquat (P²⁺) on NADPH iron-dependent lipid peroxidation (basal peroxidation) either in the presence of NADPH or in the presence of NADPH-generating systems. When NADPH is present, P²⁺ potentiates NADPH iron-dependent lipid peroxidation, but use of NADPH-generating systems cancels this effect. This may be attributed to certain components in NADPH-generating systems such as glucose-6-phosphate and sodium isocitrate, which act as iron chelators. The binding of iron by these molecules facilitates its reduction and enhances its reactivity toward dioxygen molecules, leading to the formation of reactive species capable of initiating lipid peroxidation, such as Fe³⁺-O ₂ ⁻ . Under these conditions of rapid basal peroxidation, any additional reduction of iron(III) by a reduced form of P²⁺ (P^+.) has no apparent effect on the peroxidation itself, probably because the initial reaction between iron(II) and O₂ followed by initiation of the peroxidation are both rate-limiting steps in the process. Consequently, any alteration of the composition of the reacting mixture (e.g., buffers or the generating system) must be taken into consideration because the formation of new iron chelates can change the rate of basal peroxidation and will modify the effect of redoxcycling molecules.     

The yeast FET5 gene encodes a FET3 -related multicopper oxidase implicated in iron transport

The yeast FET3 gene encodes an integral membrane multicopper oxidase required for high-affinity iron uptake. The FET4 gene encodes an Fe(II) transporter required for low-affinity uptake. To identify other yeast genes involved in iron uptake, we isolated genes that could, when overexpressed, suppress the iron-limited growth defect of a fet3 fet4 mutant. The FET5 gene was isolated in this screen and it encodes a multicopper oxidase closely related to Fet3p. Several observations indicate that Fet5p plays a role analogous to Fet3p in iron transport. Suppression of the fet3 fet4 mutant phenotype by FET5 overexpression required the putative FTR1 transporter subunit of the high-affinity system. Fet5p is an integral membrane protein whose oxidase domain is located on the cell surface or within an intracellular compartment. Oxidase activity measured in cells with altered levels of FET5 expression suggested that Fet5p is a functional oxidase. FET5 overexpression increased the rate of iron uptake by a novel uptake system. Finally, FET5 mRNA levels are regulated by iron and are increased in cells grown in iron-limited media. These results suggest that Fet5p normally plays a role in the transport of iron. Received: 12 May 1997 / Accepted: 4 July 1997     

A novel biofuel cell harvesting energy from activated human macrophages

Macrophage phagocytosis activates NADPH oxidase, an electron transport system in the plasma membrane. This study examined the feasibility of utilizing electrons transferred through the plasma membrane via NADPH oxidase to run a biofuel cell. THP-1 human monocytic cells were chemically stimulated to differentiate into macrophages. Further they were activated to induce a phagocytic response. During differentiation, cells became adherent to a plain gold electrode which was used as anode in a two-compartment fuel cell system. The current production in the fuel cell always corresponded to the NADPH oxidase activity, which was evaluated by the amount of superoxide anion produced upon stimulation in combination with the expression levels of the different NADPH oxidase subunits in cells. Moreover, our results of different inhibitory tests let us conclude that (i) the current observed in the fuel cell originates from NADPH oxidase in activated macrophages and (ii) there are multiple electron transport pathways from the cells to the electrode. One pathway involves superoxide anions produced upon stimulation, additional not yet identified electron transport occurs independently of superoxide anions.This type of novel biofuel cell driven by living human cells may eventually develop into a battery replacement for small medical devices.     

5-Lipoxygenase plays an essential role in 4-HNE-enhanced ROS production in murine macrophages via activation of NADPH oxidase

Abstract

4-Hydroxynonenal (HNE) mediates oxidative stress-linked pathological processes; however, its role in the generation of reactive oxygen species (ROS) in macrophages is still unclear. Thus, this study investigated the sources and mechanisms of ROS generation in macrophages stimulated with HNE. Exposure of J774A.1 cells to HNE showed an increased production of ROS, which was attenuated by NADPH oxidase as well as 5-lipoxygenase (5-LO) inhibitors. Linked to these results, HNE increased membrane translocation of p47phox promoting NADPH oxidase activity, which was attenuated in peritoneal macrophages from 5-LO-deficient mice as well as in J774A.1 cells treated with a 5-LO inhibitor, MK886 or 5-LO siRNA. In contrast, HNE-enhanced 5-LO activity was not affected by inhibition of NADPH oxidase. Furthermore, leukotriene B₄, 5-LO metabolite, was found to enhance NADPH oxidase activity in macrophages. Altogether, these results suggest that 5-LO plays a critical role in HNE-induced ROS generation in murine macrophages through activation of NADPH oxidase.     

Systemic upregulation of NADPH oxidase in diet-induced obesity in rats

Abstract

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is upregulated in a variety of tissues in obesity. It is still unclear as to whether NADPH oxidase upregulation in a specific tissue is part of a systemic response. Here we analyzed the expression pattern of NADPH oxidase in vascular, adipose, and kidney tissues in a rat model of diet-induced obesity. After weaning, rats were fed either a normal or high-fat diet for 12 weeks. The high-fat diet resulted in 20% increased body weight. In the aorta, Nox4 expression was increased by three-fold in obese rats. Upregulations of p22phox and p47phox in adipose, and Nox4, p22phox, and p47phox in kidney were observed in obesity. Marked increases in plasma leptin and insulin were observed, with more modest changes in adiponectin in obese rats. The average systolic blood pressure in the obese group was 11 mmHg higher than that of lean rats (P < 0.005). There was a significant correlation between blood pressure and aortic Nox4 expression (P < 0.01). In cultured vascular smooth muscle cells, adiponectin reduced the expression of Nox4 in a protein kinase A-dependent manner. Our results suggest that upregulation of NADPH oxidase in multiple tissues during obesity appears to be a systemic response. At least in vitro, adiponectin may have a protective antioxidant role by suppressing vascular NADPH oxidase expression. The association between NADPH oxidase Nox4 expression in the vasculature and the elevated blood pressure in obesity requires further investigation.     

Effect of ferrous ions on microsomal phospholipid peroxidation and related light emission

Purified NADPH cytochrome c reductase catalyzes the oxidation of NADPH in the presence of Fe³⁺, ADP and EDTA. EDTA in this system appears to elevate the redox potential of ferric ion and of its iron complex thereby facilitating the transfer of one electron from NADPH to tri-valent iron (more rapidly than superoxide is formed) through a flavin moiety in the reductase, but it diminishes the concentration of free iron to be required for phospholipid peroxidation.

The reduction of Fe³⁺ by the xanthine-xanthine oxidase system is different from that manifested by the NADPH-NADPH cytochrome c reductase system in the manner in which the former is carried out in the main by O_staggered2^staggered− · generated by the substrate - O₂ - enzyme interaction.

Reduced iron, which is free in the solution, plays an important role for the initiation and propagation of the phospholipid peroxidation, monitored by malondialdehyde assay and light emission.

In the xanthine-induced lipoxygenation system, the ·OH radical, probably produced from hydrogen peroxide by the action of Fe²⁺, is not involved in the initiation of the peroxidative cleavage of phospholipid in microsomal lipoprotein.     

Lipid peroxide formation in microsomes. The role of non-haem iron

1. Metal ion-chelating agents such as EDTA, o-phenanthroline or desferrioxamine inhibit lipid peroxide formation when rat liver microsomes prepared from homogenates made in pure sucrose are incubated with ascorbate or NADPH. 2. Microsomes treated with metal ion-chelating agents do not form peroxide on incubation unless inorganic iron (Fe²⁺ or Fe³⁺) in a low concentration is added subsequently. No other metal ion can replace inorganic iron adequately. 3. Microsomes prepared from sucrose homogenates containing EDTA (1mm) do not form lipid peroxide on incubation with ascorbate or NADPH unless Fe²⁺ is added. Washing the microsomes with sucrose after preparation restores most of the capacity to form lipid peroxide. 4. Lipid peroxide formation in microsomes prepared from sucrose is stimulated to a small extent by inorganic iron but to a greater extent if adenine nucleotides, containing iron compounds as a contaminant, are added. 5. The iron contained in normal microsome preparations exists in haem and in non-haem forms. One non-haem component in which the iron may be linked to phosphate is considered to be essential for both the ascorbate system and NADPH system that catalyse lipid peroxidation in microsomes.     

Anti-inflammatory action of gentamycin through inhibitory effect on neutrophil NADPH oxidase activity

The effects of gentamycin on the NADPH oxidase (EC 1.6.99.6) from human neutrophils in both whole-cell and fully soluble (cell-free) systems were investigated. Gentamycin was found to inhibit, concentration-dependently, the superoxide generation of neutrophils exposed to phorbol myristate acetate in a whole-cell system and the activation of superoxide-generating NADPH oxidase by sodium dodecyl sulfate in a cell-free system. The concentrations of the drug required for 50% inhibition of the oxidase (IC₅₀) were 150 μM in the whole-cell system and 10 μM in the cell-free system. In addition, in the cell-free system, the drug did not change the K_m value for NADPH of the oxidase. However, gentamycin did not the superoxide generation of NADPH oxidase after its activation in the cell-free system, suggesting that the drug do not have superoxide-scavenger action. These results suggest that gentamycin, an aminoglycoside antibiotic, may exhibit an anti-inflammatory action due to inhibition of neutrophil NADPH oxidase activation.     

Cloning of putative subunits of the soybean plasma membrane NADPH oxidase involved in the oxidative burst by antibody expression screening

Summary Plant cells respond to a variety of external signals with the production of reactive-oxygen species. The enzyme system generating these reactive-oxygen species is believed to be an NADPH oxidase located in the plasma membrane and sharing similarities with the NADPH oxidase from mammalian macrophages. Antibodies directed against individual subunits (p22 ^phox , p47 ^phox , p67 ^phox ) of the human NADPH oxidase cross-react with soybean proteins of a similar size and subcellular location. An extensive expression screening of a soybean cDNA-library with the anti-human NADPH oxidase antibodies gave a single class of cDNA-clones for each antibody. However, the sequence analysis of these clones clearly demonstrates that the different antibodies recognise proteins which are unrelated to the expected oxidase subunits. The anti-p22 ^phox antibody recognised a microsomal protein with no significant homology to any known protein in the database. One anti-p47 ^phox antibody cross-reacted with the UDP-glucose dehydrogenase and another antibody bound to the chaperon peptidyl prolyl-cis-trans isomerase, both soluble cytosolic proteins. The anti-p67 ^phox antibody detected the soluble enzyme acetohydroxy acid reductoisomerase. Chromatography of soybean protein extracts on an ion-exchange column (MonoQ, FPLC) gave a perfect comigration of the enzyme activity with the antibody signal, thus confirming these unexpected results by independent biochemical experiments.Abbreviations AARI acetohydroxy acid reductoisomerase - DPI diphenylene iodonium - GST glutathione-S-transferase - phox NADPH oxidase of phagocytes - ROS reactive-oxygen species     

Studies on lipid peroxidation in normal and tumour tissues. The Novikoff rat liver tumour.

A study has been made of the factors that contribute to the decreased rates of lipid peroxidation under different pro-oxidant conditions in intact Novikoff tumour cells, and in microsomal suspensions prepared from Novikoff tumour cells, compared with isolated normal rat hepatocytes and microsomal suspensions prepared from normal rat liver. The pro-oxidant conditions were the addition of either NADPH, NADPH + ADP + iron, NADPH + CCl4 or ascorbate+iron to the experimental systems used, or exposure to gamma-radiation. Contributory factors to the lower rates of lipid peroxidation observed include: a significant decrease in the polyunsaturated fatty acid content of Novikoff cells or Novikoff microsomes; the decreases are especially marked for the C20:4 and C22:6 fatty acids; a very marked reduction in NADPH-cytochrome c reductase; and no detectable content of cytochrome P-450. Another, and in our opinion critical, contribution to the diminished rate of lipid peroxidation in the tumour material is the substantial increase in alpha-tocopherol relative both to total lipid and to methylene-interrupted double bonds in fatty acids. Moreover, the alpha-tocopherol is the major contributor to lipid-soluble chain-breaking antioxidant in lipid extracts of normal liver and of Novikoff tumour material.     

Bovine heart microsomes contain an Mr = 66,000 non-heme iron protein which stimulates NADPH oxidation

Bovine heart microsomes have been found to contain a non-heme iron protein which serves as an electron acceptor for NADPH-cytochrome P-450 reductase and therefore stimulates NADPH oxidation. This protein, tentatively referred to as Microsomal Iron Protein (MIP), has been extracted with Triton N-101 and purified by ion exchange chromatography on CM- and DEAE-celluloses and gel filtration on Sepharose 6B. MIP is an Mr = 66,000 monomer with 17 atoms of Fe(III)/molecule. Incubation with dithionite removes iron from MIP and abolishes the stimulation of NADPH oxidation, but subsequent incubation with nitrilotriacetic-Fe(III) reincorporates iron and restores the stimulation of NADPH oxidation. Oxygen is the ultimate electron acceptor. In the presence of oxygen, the enzymatic reduction of MIP Fe(III) is followed by the reoxidation of Fe(II) at the expense of oxygen, generating superoxide anion and regenerating MIP Fe(III) for the continuous oxidation of NADPH. In the absence of oxygen, electron transfer from the reductase to MIP Fe(III) causes the release of Fe(II), which limits the ability of MIP to serve as an electron acceptor and stimulate NADPH oxidation. The--NH2-terminal of MIP has been sequenced, and no homology has been found with the sequence of other iron storage or transport proteins such as ferritin or transferrin.