External NADPH Research Articles

Rotenone-insensitive NAD(P)H dehydrogenases in plants: Immunodetection and distribution of native proteins in mitochondria

Antisera produced against peptides deduced from potato nda1 and ndb1, homologues of yeast genes for mitochondrial rotenone-insensitive NADH dehydrogenases, recognise respective proteins upon expression in Escherichia coli. In western blots of potato ( Solanum tuberosum L.) mitochondrial proteins, the NDB and NDA antibodies specifically detect polypeptides of 61 and 48 kDa, respectively. The proteins are found in mitochondria of flowers, leaves and tubers. Different signal intensities are seen relative to other respiratory chain components when organs are compared, indicating variations in relative abundance of dehydrogenases within the plant. The antibodies detect single polypeptides, of similar size as in potato, in mitochondria from several plant species. No specific cross-reaction was found in chloroplasts, but a weak NDA signal of 50 kDa was found in microsomes, possibly associated with peroxisomes. Two-dimensional native/SDS-PAGE analyses indicate that both NDA and NDB proteins reside as higher molecular mass forms, possibly oligomeric. The NDB immunoreactive protein is released by sonication of mitochondria, but is resistant to extraction by digitonin and partially to Triton X-100. In comparison, the NDA protein remains bound to the inner membrane at sonication or digitonin treatment, but can be solubilised with Triton. Investigation of a beetroot ( Beta vulgaris L.) induction system for external NADH dehydrogenase indicates that the NDB antibody does not recognise the induced external NADH dehydrogenase in this species, but possibly an external NADPH dehydrogenase.

The External Calcium-dependent NADPH Dehydrogenase from Neurospora crassa Mitochondria

We have inactivated the nuclear gene coding for a putative NAD(P)H dehydrogenase from the inner membrane of Neurospora crassa mitochondria by repeat-induced point mutations. The respiratory rates of mitochondria from the resulting mutant (nde-1) were measured, using NADH or NADPH as substrates under different assay conditions. The results showed that the mutant lacks an external calcium-dependent NADPH dehydrogenase. The observation of NADH and NADPH oxidation by intact mitochondria from the nde-1 mutant suggests the existence of a second external NAD(P)H dehydrogenase. The topology of the NDE1 protein was further studied by protease accessibility, in vitro import experiments, and in silico analysis of the amino acid sequence. Taken together, it appears that most of the NDE1 protein extends into the intermembrane space in a tightly folded conformation and that it remains anchored to the inner mitochondrial membrane by an N-terminal transmembrane domain.

Identification of the site where the electron transfer chain of plant mitochondria is stimulated by electrostatic charge screening

Modular kinetic analysis was used to determine the sites in plant mitochondria where charge-screening stimulates the rate of electron transfer from external NAD(P)H to oxygen. In mitochondria isolated from potato (Solanum tuberosum L.) tuber callus, stimulation of the rate of oxygen uptake was accompanied by a decrease in the steady-state reduction level of coenzyme Q, and by a small decrease in the steady-state reduction level of cytochrome c. Modular kinetic analysis around coenzyme Q revealed that stimulation of the rate was due to stimulation of quinol oxidation via the cytochrome pathway (cytochrome bc1, cytochrome c and cytochrome c oxidase). It was not a consequence of any effect on quinone reduction (by external NADH or NADPH dehydrogenase). This explains the salt-induced decrease in the steady-state reduction level of coenzyme Q. Analysis around cytochrome c revealed that stimulation by salts was due to a dual effect on the respiratory chain. The kinetic curves for the oxidation and reduction pathways of cytochrome c revealed that they were both activated by salt, the simultaneity explaining the small variation observed in the steady-state reduction level of cytochrome c. A simple kinetic core model is used to show that changes in the rate of dissociation of cytochrome c from the membrane can explain the observed kinetic changes in both cytochrome c reduction and cytochrome c oxidation. The stimulation is proposed to be the result of an increase in the rate constant of cytochrome c dissociation from the membrane induced by cation screening. We conclude that this type of modular kinetic analysis is a powerful tool to identify and quantitatively characterize multiple-site effects on the mitochondrial respiratory chain.

Development of a new genotoxicity test system with Salmonella typhimurium OY1001/1A2 expressing human CYP1A2 and NADPH– P450 reductase

In order to develop a new tester strain detecting environmental promutagens and procarcinogens, we introduced two plasmids into Salmonella typhimurium TA1535; one contains the cDNAs of human cytochrome P450 ( P450 or CYP) 1A2 and NADPH– P450 reductase and the other (pOA101) a umuC″lacZ fusion gene. The newly developed tester strain, S. typhimurium OY1001/1A2, was found to express P450 at a level of 0.15 nmol/ml in whole cell culture. Membrane fractions, when isolated from this tester strain, contained 0.04 P450 nmol/mg protein and a reductase activity of 170 nmol cytochrome c reduced/min/mg protein and were active in catalyzing CYP1A2-dependent 7-ethoxyresorufin O-deethylation and metabolic activation of heterocyclic aromatic amines to DNA-damaging products in a conventional tester S. typhimurium NM2009 strain, only when NADPH was added as a reducing equivalent. In the OA1002/1A2 strain, heterocyclic aromatic amines (e.g., IQ, MeIQ, and MeIQx) were found to be activated to reactive metabolites that cause induction of umuC gene expression in a dose-dependent manner, without addition of external NADPH. These results indicate that the newly established strain can be of use to detect mutagenic and carcinogenic potencies of environmental chemicals without addition of metabolic activation system.

Cobalt and desferrioxamine reveal crucial members of the oxygen sensing pathway in HepG2 cells

Cobalt and desferrioxamine, like hypoxia, stimulate the production of erythropoietin in HepG2 cells. It is believed that cobalt as well as desferrioxamine interact with the central iron atom of heme proteins by changing their redox state similar to hypoxia. A subsequent decrease of the intracellular H2O2 levels under hypoxia was presumed to be the key event for stimulating erythropoietin production. We therefore investigated whether cobalt and desferrioxamine control the intracellular H2O2 levels that regulate gene expression by interacting with hemeproteins. Deconvolution of light absorption spectra revealed respiratory heme proteins such as cytochrome c, b558 and cytochrome aa3, as well as cytochrome b558, which is a nonrespiratory heme protein found in HepG2 cells. Whereas respiratory heme proteins are located in mitochondria, cytochrome b558 similar to the one described for the neutrophil NADPH oxidase can be visualized in the cell membrane of HepG2 cells by immunohistochemistry. Incubation with cobalt (100 microM/24 hr) interacts predominantly with cytochrome b558 and cytochrome b558. The interaction of cobalt with the respiratory chain results in an increased oxygen consumption of HepG2 cells as revealed by PO2 microelectrode measurements. Desferrioxamine (130 microM/24 hr), however has no influence on the cytochromes. In response to an external application of NADH (1 mM), the membrane bound cytochrome b558 produces two times more O2- than to the external NADPH (1 mM) application. Neither desferrioxamine not cobalt has any influence on the NADH stimulated O2- generation. Incubation with cobalt or with desferrioxamine, however, leads to a decrease of the intracellular H2O2 level as revealed by the dihydrorhodamine 123 technique, perhaps causing the well-known enhanced erythropoietin production. The cobalt-induced H2O2 decrease seems to be caused by an increased activity of the glutathion peroxidase that is also induced under hypoxia. Desferrioxamine, however, leads to an apparent H2O2 decrease only because it seems to inhibit the iron catalyzed reaction of H2O2 with dihydrorhodamine 123, hinting at the occurrence of the Fenton reaction in HepG2 cells. Therefore, it must be determined whether or not degradation products of H2O2 by the Fenton reaction suppress erythropoietin production under normoxia.

Identification of an external NADH oxidase in rat kidney cortex mitochondria.

Intact rat kidney cortex mitochondria oxidize external NADH and NADPH. Basal NADH oxidation of mitochondria, but not basal NADPH oxidation, is stimulated by hexammine-ruthenium (HR). 10.7 mumol/l HR induce 50% of the maximal NADH-oxidizing activity and amounted to 169 nmol NADH/min/mg of mitochondrial protein. The HR-stimulated NADH oxidation involves a stoichiometric 1:1 oxygen consumption. The electron transfer from NADH to oxygen does not occur via the respiratory chain complex I, but is connected with a superoxide anion radical formation. Experiments with intact mitochondria, submitochondrial particles and inner mitochondrial membranes show that rat kidney cortex mitochondria possess a NADH oxidase localized on the outer surface of the inner mitochondrial membrane.

Oxidation of External NAD(P)H by Jerusalem Artichoke (Helianthus tuberosus) Mitochondria

The functional interaction between the externally located NAD(P)H dehydrogenase and the Q-pool acceptor site(s) in Percoll-purified mitochondria from Jerusalem artichoke (Helianthus tuberosus L. cv OB1) mitochondria has been investigated. Oxidation of exogenous NADH is stimulated by ubiquinone (UQ(1)) with a parallel decrease of the apparent K(m) for NADH. In the presence of saturating amounts of UQ(1) as electron acceptor, the K(m) (NADH) is not affected by variations of the ionic strength. Conversely, the K(m) for UQ(1) is decreased by the screening effect of negative charges on the outer membrane surface. Under low-ionic strength, the hydroxyflavone platanetin progressively inhibits NADH oxidation with a mean inhibition dose of approximately 3 nanomoles of inhibitor per milligram of protein. Interestingly, under high-ionic strength, oxidation of NADH proceeds through two platanetin binding sites, one of which has a lower affinity for the inhibitor (mean inhibition dose = 20 nanomoles per milligram protein), because it is located near the outer surface of the membrane. This latter site is the one involved in the oxidation of external NADPH and, possibly, also affected by spermine and spermidine. Similarly to NADH, oxidation of NADPH is fully sensitive to micromolar concentrations of free Ca(2+) ions; in addition, similar concentrations of the sulfhydryl reagent mersalyl are required to inhibit both NADH and NADPH oxidative activities. The results are interpreted as evidence for the presence of a single nonspecific NAD(P)H dehydrogenase.

Respiration of pea leaf mitochondria and redox transfer between the mitochondrial and extramitochondrial compartment

In this report, properties of isolated mitochondria from pea leaves have been studied in view to their function in photosynthesis metabolism of a leaf cell. (1) The rates of respiration with various substrates and with a combination of these substrates have been measured with isolated mitochondria. The highest respiration rate was found with NADH, followed by NADPH, malate and glycine. For the oxidation of NADH, NADPH, malate, glycine and 2-oxoglutarate the apparent K m values were determined. The oxidation of malate and glycine occurred independently of each other as long as electron transport was not limiting. (2) The maximal capacity of mitochondrial ATP synthesis in a leaf was estimated as about 25% of the rate of noncyclic photophosphorylation at maximal rate of photosynthesis. (3) From measurements of NADH/NAD ratios in isolated mitochondria and from previous determinations of the NADH/NAD ratio in the cytosol of spinach leaves it is discussed that in a leaf cell the NADH/NAD ratio in the mitochondrial matrix is higher than in the cytosol. (4) A comparison of the apparent K m values obtained for the oxidation of external NADH and NADPH with the corresponding concentrations found in the cytosol of spinach leaves suggests that in a leaf cell NADPH is oxidized by mitochondria at a much higher rate than NADH. (5) From the measurement of mitochondrial respiration with glycine and malate as substrates in the presence of a defined malate / oxaloacetate ratio the function of a malate-oxaloacetate shuttle is demonstrated. It is furthermore shown that a malate-aspartate shuttle does not play any significant role in redox transfer under physiological conditions.

NADP-Utilizing Enzymes in the Matrix of Plant Mitochondria

Purified potato tuber (Solanum tuberosum L. cv Bintie) mitochondria contain soluble, highly latent NAD(+)- and NADP(+)-isocitrate dehydrogenases, NAD(+)- and NADP(+)-malate dehydrogenases, as well as an NADPH-specific glutathione reductase (160, 25, 7200, 160, and 16 nanomoles NAD(P)H per minute and milligram protein, respectively). The two isocitrate dehydrogenase activities, but not the two malate dehydrogenase activities, could be separated by ammonium sulfate precipitation. Thus, the NADP(+)-isocitrate dehydrogenase activity is due to a separate matrix enzyme, whereas the NADP(+)-malate dehydrogenase activity is probably due to unspecificity of the NAD(+)-malate dehydrogenase. NADP(+)-specific isocitrate dehydrogenase had much lower K(m)s for NADP(+) and isocitrate (5.1 and 10.7 micromolar, respectively) than the NAD(+)-specific enzyme (101 micromolar for NAD(+) and 184 micromolar for isocitrate). A broad activity optimum at pH 7.4 to 9.0 was found for the NADP(+)-specific isocitrate dehydrogenase whereas the NAD(+)-specific enzyme had a sharp optimum at pH 7.8. Externally added NADP(+) stimulated both isocitrate and malate oxidation by intact mitochondria under conditions where external NADPH oxidation was inhibited. This shows that (a) NADP(+) is taken up by the mitochondria across the inner membrane and into the matrix, and (b) NADP(+)-reducing activities of malate dehydrogenase and the NADP(+)-specific isocitrate dehydrogenase in the matrix can contribute to electron transport in intact plant mitochondria. The physiological relevance of mitochondrial NADP(H) and soluble NADP(H)-consuming enzymes is discussed in relation to other known mitochondrial NADP(H)-utilizing enzymes.

Hydrogen peroxide production by monoamine oxidase in isolated rat-brain mitochondria: its effect on glutathione levels and Ca 2+ efflux

H 2O 2 production and accumulation during incubation of isolated rat-brain mitochondria with substrates of monoamine oxidase A and B were investigated. All substrates gave rise to an accumulation of H 2O 2 which was inhibited by malate+pyruvate or isocitrate, consistent with a need for mitochondrial NADPH to maintain glutathione in the reduced state. However, in the absence of these additions the level of reduced glutathione decreased only by about 30%, indicating that only a fraction of the mitochondrial glutathione pool was accessible to the glutathione peroxidase and glutathione reductase activities responsible for the continuous removal of H 2O 2 generated by momoamine oxidase. The ,H 2O 2 accumulation was also inhibited by externally added reduced glutathione or NADPH but not NADH. External NADPH was oxidized by added oxidized glutathione but not α-ketoglutarate + NH 4 +. These results suggest that the removal of H 2O 2 generated by monoamine oxidase proceeds by way of special fractions of glutathione peroxidase and glutathione reductase that are located in the intermembrane space of mitochondria in such a way that they can react with both intra- and extra-mitochondrial glutathione and NADPH, possibly at the contact sites between the inner and outer mitochondrial membranes. Evidence is also presented that H 2O 2 generated by monoamine oxidase enhances Ca 2+ release from mitochondria and may thus function as a regulator of mitochondrial Ca 2+ efflux.

Tightly bound NADPH in Proteus mirabilis PR catalase

Previous work with catalase from Proteus mirabilis PR, a mutant with high resistance to H2O2, had shown that two different forms of this enzyme, named A and B, could be resolved by anion exchange chromatography. The present results showed that catalase B differed from A by the presence of bound NADPH. Direct evidence for the presence of this dinucleotide within the bacterial catalase was obtained using high performance liquid chromatography. The obtention of pure catalase B without any addition of external NADPH showed that this dinucleotide was bound tightly to the protein as to be carried along with the protein during purification. Catalase complex II was formed from either-enzyme A or B by the continuous supply of low amounts of hydrogen peroxide as in the case of bovine liver catalase. The reversal of complex II formation from catalase B was obtained using a NADPH-regenerating system (isocitrate plus isocitrate dehydrogenase) in the absence of any added dinucleotide, confirming that this cofactor was present in the enzyme molecule. The addition of dinucleotide was required when performing the same experiment with catalase A. This was the first observation of NADPH binding by a bacterial catalase, and the results agreed with the former theory where NADPH is considered as a rescuer of the enzyme from inactivation in the form of complex II.Key words: catalase, bovine liver catalase, NADPH, Proteus mirabilis.

Oxidation of reduced nicotinamide hypoxanthine dinucleotide phosphate by intact rat liver mitochondria.

1. While intact rat liver mitochondria oxidize external NADPH at an extremely low rate (0.1–0.4 nmole per mg protein per min), reduced nicotinamide hypoxanthine dinucleotide phosphate (NHDPH) is relatively rapidly oxidized (12–13 nmoles per mg protein per min). 2. After sonication, the rate of mitochondrial NHDPH oxidation is increased by 2-fold, and the NADPH oxidation rate is double that of NHDPH. 3. NADPH and NHDPH oxidation is neither coupled to ATP synthesis nor inhibited by rotenone or CN−, but is inhibited under anaerobic conditions. These data indicate that, although molecular oxygen is the ultimate electron acceptor, the respiratory chain is not functional in the oxidation reaction. 4. The NADPH oxidase activity resides in the submitochondrial particle fraction isolated from sonicated mitochondrial preparations. 5. These results are interpreted to indicate the presence of an NADPH oxidase system in rat liver mitochondria which is bound to the inner surface of the inner membrane and independent of the respiratory chain. Further, it is concluded that the inner membrane may be significantly more permeable to NHDPH than to NADPH.