The role of NADH oxidase in growth and physical membrane displacement

Summary An NADH oxidase activity of the plasma membrane that was growth factor- and hormone-stimulated has been reported and characterized extensively. The activity is constitutively activated in cancer. Thiol groups are important to its activity and its activity as a thiol oxidoreductase or thiol interchange activity may be important to its functioning in growth and membrane trafficking. The activity is inhibited in the presence of GDP and brefeldin A and appears to be found not only at the plasma membrane but also with the Golgi apparatus. The phenotype of an inhibited NADH oxidase is a failure of cells to enlarge. The hypothesis under investigation is that the hormone- and growth factor-stimulated NADH (quinone)/thiol oxidase functions as an essential component of physical membrane displacements associated with cell elongation, vesicle budding and pleomorphic membrane changes in both animal and plant cells.     

Removal of ferredoxin:NADP+ oxidoreductase from thylakoid membranes, rebinding to depleted membranes, and identification of the binding site

Ferredoxin-NADP+ oxidoreductase associates with thylakoid membranes into two pools of different binding strength that are experimentally distinguished on the basis of resistance to removal by washes in low ionic strength media. The nondenaturing zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid is uniquely able to remove the more tightly bound pool of enzyme, without solubilization of major membrane proteins. The reconstitution of reductase onto depleted thylakoid membranes requires available membrane binding sites and cations, in order of effectiveness trivalent greater than divalent greater than monovalent. The hetero/bifunctional 125I-iodinated Denny-Jaffe cross-linking reagent yields a 54-kDa, covalently cross-linked adduct between ferredoxin-NADP+ oxidoreductase and a component of the thylakoid membrane. Our results show that the more tightly bound pool of enzyme is associated with the 17.5-kDa reductase-binding protein (Vallejos, R. H., Ceccarelli, E., and Chan, R. (1984) J. Biol. Chem. 259, 8048-8051).     

Detection and isolation of the NADPH-binding protein of the NADPH:O2 oxidoreductase complex of human neutrophils

Neutrophils assayed with nitro blue tetrazolium (NBT) exhibit intracellular rather than extracellular superoxide-generating activity when stimulated with phorbol myristate acetate. Enzyme activity is stimulated by anionic detergents, reversibly inhibited by 2',3'-NADPH dialdehyde, and present in equal levels in membrane fractions obtained from phorbol myristate acetate-stimulated and resting cell suspensions. Solubilized membrane shows enzyme activity co-eluting on molecular sieving columns with the cytochrome b redox component of the oxidoreductase complex. Enzyme activity was resolved free of the cytochrome b component following passage of solubilized membrane extracts through QAE-Sephadex anion exchange columns. Enzyme activity measured by the NBT assay appears to be that associated with the NADPH binding protein of the oxidoreductase complex. When exposed to NBT and NADPH this component of the oxidoreductase generates superoxide independent of cytochrome b.     

Calnexin,calreticulin, and ERp57 cooperate in disulfide bond formation in human CD1d heavy chain

Members of the CD1 family of membrane glycoproteins can present antigenic lipids to T lymphocytes. Like major histocompatibility complex class I molecules, they form a heterodimeric complex of a heavy chain and beta(2)-microglobulin (beta(2)m) in the endoplasmic reticulum (ER). Binding of lipid antigens, however, takes place in endosomal compartments, similar to class II molecules, and on the plasma membrane. Unlike major histocompatibility complex class I or CD1b molecules, which need beta(2)m to exit the ER, CD1d can be expressed on the cell surface as either a free heavy chain or associated with beta(2)m. These differences led us to investigate early events of CD1d biosynthesis and maturation and the role of ER chaperones in its assembly. Here we show that CD1d associates in the ER with both calnexin and calreticulin and with the thiol oxidoreductase ERp57 in a manner dependent on glucose trimming of its N-linked glycans. Complete disulfide bond formation in the CD1d heavy chain was substantially impaired if the chaperone interactions were blocked by the glucosidase inhibitors castanospermine or N-butyldeoxynojirimycin. The formation of at least one of the disulfide bonds in the CD1d heavy chain is coupled to its glucose trimming-dependent association with ERp57, calnexin, and calreticulin.     

A complex II defect affects mitochondrial structure,leading to ced-3- and ced-4-dependent apoptosis and aging

The mev-1(kn1) mutation of Caenorhabditis elegans is in Cyt-1, which encodes a subunit of succinate-coenzyme Q oxidoreductase in the mitochondrial electron transport chain. Mutants are hypersensitive to oxidative stress and age precociously in part because of increased superoxide anion production. Here, we show that mev-1 mutants are defective in succinate-coenzyme Q oxidoreductase, possess ultrastructural mitochondrial abnormalities (especially in muscle cells), show a loss of membrane potential, have altered CED-9 and Cyt-1 protein levels under hyperoxia, and contain ced-3-and ced-4-dependent supernumerary apoptotic cells. These defects likely explain the failure of mev-1 to complete embryonic development under hyperoxia as well as its reduced life span.     

Purification and resolution of NADH diaphorase activity from NADPH diaphorase-linked: O2 oxidoreductase activity of human neutrophils

Intrinsic NADPH diaphorase activity is a component of the membrane-bound NAD(P)H:O2 oxidoreductase of human neutrophils. NADH-specific diaphorase activity is also present in membrane fractions rich in oxidoreductase activity. Studies were undertaken to determine whether the NADH diaphorase might also be intrinsic to the oxidoreductase. The latter diaphorase was freed from the membrane by detergent extraction and partially purified approximately 80-fold. Its apparent molecular weight following solubilization in deoxycholate and Tween-20 was 204 000 +/- 10 000. The specific activity of the partially purified diaphorase with ferricyanide as electron acceptor was 7.6 X 10(3) mU/mg protein, its pH optimum was 7.0, and its Km for NADH was 13 microM. It is completely devoid of NADPH diaphorase activity, lacks the capacity to reduce molecular oxygen, yet readily reduces ferricyanide, 2,6-dichlorophenolindophenol and ferricytochrome c. Whereas the NADH diaphorase was freed from the particulate fraction of cell lysates by extraction in 10 mM Tris-HCl buffer (pH 8.6) made up in 15% glycerol and 0.5% Tween-20, NADPH-dependent diaphorase and superoxide-generating activities also present in the membrane were not. These observations make it unlikely that the principal membrane-bound NADH diaphorase found in human neutrophils is a component of the NAD(P)H:O2 oxidoreductase, despite its common association in the same particulate fraction of cell lysates.     

Masking of Bacillus megaterium KM membrane reduced nicotinamide adenine dinucleotide oxidase and solubilization studies

Solubilization of a reduced nicotinamide adenine dinucleotide (NADH)-2,6 dichlorophenol indophenol (DCIP) oxidoreductase associated with the membrane NADH oxidase system of Bacillus megaterium KM was effected by treatment with 0.2% sodium deoxycholate, 8 m urea, or buffer (pH 9.0) in the presence of ethyl-enediaminetetraacetate. These treatments inactivated membrane NADH oxidase. It was found that membrane NADH oxidase and NADH-DCIP oxidoreductase were masked in membranes. Several procedures, including brief sonic oscillation, treatment with 0.05% deoxycholate, prolonged stirring at 4 C with 10% glycerol, and washing in the absence of Mg(2+), unmasked the oxidase and oxidoreductase activities. It was necessary to study the masking and unmasking of these activities to quantitate adequately the effects of solubilization procedures. Further information on the localization of oxidase and oxidoreductase in subcellular fractions and the effects of electron transport inhibitors on NADH oxidation was also obtained.     

Investigations on microbial sulfur respiration. Isolation, purification, and characterization of cellular components from Spirillum 5175.

The sulfur-reducing bacterium Spirillum 5175 was investigated with regard to membrane constituents that might be part of the sulfur oxidoreductase which converts elemental sulfur to hydrogen sulfide. Regardless of the electron acceptor used for cultivation of the bacteria, i.e. elemental sulfur, fumarate, or nitrate (Sp. 5175S,F,N), the qualitative pattern of cytochromes and Fe-S proteins did not change significantly, as documented by ultraviolet/visible and electron paramagnetic resonance spectroscopy of oxidized (as isolated) and reduced (dithionite) samples. With elemental sulfur the prominent cytochrome exhibited absorption maxima at 553, 522.5 and 426 nm in the reduced state. In fumarate-grown cells two prominent cytochromes were found with maxima at 561, 551, 530, 521 and 430 nm. Two b-type cytochromes with Em at -198 mV and -20 mV vs the standard hydrogen electrode were identified in the membrane fraction of Sp. 5175F. A yellow pigment was extracted and identified as a flexirubin-type pigment. Although present in large quantities, it seemed not to be involved in the reduction of elemental sulfur. Menaquinone, MK 6 (Mr 580) was the prominent quinone identified in Sp. 5175. Characterization of a second quinone was not attempted because of its much lower concentration. The membrane constituents of Sp. 5175 were solubilized by a variety of detergents and detergent mixtures. A colorimetric procedure with photochemically reduced phenosafranin as the electron donor and cysteamine trisulfide (RS-S-SR, R = -CH2CH2NH2) as the electron acceptor was used to detect sulfur oxidoreductase activity. Three membrane proteins of Sp. 5175 were purified: (1) an [NiFe] hydrogenase, homogeneous by SDS/polyacrylamide gel electrophoresis, with electron paramagnetic resonance signals as isolated at gx,y,z = 2.01, 2.16, 2.33 (100 K), and a strong signal at g = 2.02 below 20 K; (2) a cytochrome b, Fe-S-dependent fumarate reductase, and (3) a protein apparently linked to the sulfur oxidoreductase activity. In contrast to fumarate reductase, no b-type cytochrome was present in the fractions exhibiting sulfur oxidoreductase activity. The presence of Fe-S centers was demonstrated by electron paramagnetic resonance spectroscopy at 10 K. It is not clear whether the c-type cytochrome in the same fractions is part of the sulfur-reducing apparatus of Sp. 5175.     

psbG is not a photosystem two gene but may be an ndh gene

A gene of the chloroplast genome has been designated the psbG gene on the basis that in maize the gene product is a 24-kDa polypeptide of photosystem two (PS2) (Steinmetz, A. A., Castroviejo, M., Sayre, R. T., and Bogorad, L. (1986) J. Biol. Chem. 261, 2485-2488). We have located and sequenced the equivalent gene in wheat (Triticum aestivum) and have raised specific antibodies to the gene product following its expression in Escherichia coli as a beta-galactosidase fusion protein. Using these antibodies, we have investigated the location of the gene product in various thylakoid membrane fractions of pea (Pisum sativum). The gene product of apparent molecular mass 27-28 kDa is severely depleted in PS2-enriched membrane preparations and its distribution between stromal and granal regions of the membrane is distinct to that of the psbC gene product which is known to be a core polypeptide of PS2. We therefore conclude that psbG does not code for a component of PS2 but instead suggest that it is present in a novel protein complex of the thylakoid membrane. On the basis of 1) the conserved overlap between psbG and ndhC, a chloroplast gene which shows significant homology to a mitochondrial gene that codes for a subunit of the NADH-ubiquinone oxidoreductase of mitochondria, and 2) sequence similarity between the psbG gene product and the ndh gene product of E. coli, which codes for a respiratory NADH dehydrogenase, we propose that this ill-defined complex functions as a NADH or NADPH-plastoquinone oxidoreductase.     

Structure of a voltage-dependent K+ channel beta subunit.

The integral membrane subunits of many voltage-dependent potassium channels are associated with an additional protein known as the beta subunit. One function of beta subunits is to modify K+ channel gating. We have determined the structure of the conserved core of mammalian beta subunits by X-ray crystallography at 2.8 A resolution. Like the integral membrane component of K+ channels, beta subunits form a four-fold symmetric structure. Each subunit is an oxidoreductase enzyme complete with a nicotinamide co-factor in its active site. Several structural features of the enzyme active site, including its location with respect to the four-fold axis, imply that it may interact directly or indirectly with the K+ channel's voltage sensor. This structure suggests a mechanism for coupling membrane electrical excitability directly to chemistry of the cell.     

Accumulation of the pre-assembled membrane arm of NADH:ubiquinone oxidoreductase in mitochondria of manganese-limited grown Neurospora crassa.

The NADH:ubiquinone oxidoreductase (complex I) of mitochondria is constructed from two arms arranged perpendicular to each other. The peripheral arm protruding into the matrix contains the proximal section of the electron pathway, and the membrane arm with all mitochondrially encoded subunits contains the distal section of the electron pathway. When Neurospora crassa is grown under manganese limitation the formation of the peripheral arm is disturbed, but the membrane arm containing the iron-sulfur cluster N-2, is accumulated. An extra-polypeptide, assumed to be a chaperone, is found to be associated with this pre-assembled membrane arm.     

Development of a Membrane-Bound Respiratory System Prior to and During Sporulation in Bacillus cereus and Its Relationship to Membrane Structure

Bulk membrane fragments were prepared from cells of Bacillus cereus ATCC 4342 harvested at different stages of growth and sporulation and examined for enzymes involved in electron transport functions. The presence of succinate: DCPIP oxidoreductase (EC 1.3.99.1), succinate: cytochrome c oxidoreductase (EC 1.3.2.1), NADH:DCPIP oxidoreductase (EC 1.6.99.1), NADH:cytochrome c oxidoreductase (EC 1.6.2.1), succinate oxidase [succinate: (O(2)) oxidoreductase, EC 1.3.3.1], and NADH oxidase [NADH:(O(2)) oxidoreductase, EC 1.6.3.1] were demonstrated in membrane fragments from vegetative cells, early and late stationary-phase cells, and in cells undergoing sporulation. During the transition from a vegetative cell to a spore, there was a significant increase in the levels of enzymes associated with energy production via the electron transport system. Cytochromes of the a, b, and c type were detected in all membrane preparations; however, there was a marked increase in the level of cytochromes by the end of vegetative growth which remained throughout sporulation; there were no qualitative changes in the cytochromes throughout growth and sporulation. Sporulation was inhibited by cyanide, stressing the significance of the electron transport system. Enzyme activities were partially masked in washed membrane fragments; however, unmasking (stimulation) was achieved by sodium deoxycholate, sodium dodecyl sulfate, or Triton X-100. The degree of enzyme masking was less in vegetative cell membrane fragments than in membranes prepared from stationary-phase or sporulating cells. Results indicate the development of a membrane-bound electron transport system in B. cereus by the end of growth and prior to sporulation, which results in an increased masking of a number of enzymes associated with the terminal respiratory system of the cell.     

Biogenesis of a putative channel protein, ComEC, required for DNA uptake: membrane topology, oligomerization and formation of disulphide bonds

ComEC is a putative channel protein for DNA uptake in Bacillus subtilis and other genetically transformable bacteria. Membrane topology studies suggest a model of ComEC as a multispanning membrane protein with seven transmembrane segments (TMSs), and possibly with one laterally inserted amphipathic helix. We show that ComEC contains an intramolecular disulphide bond in its N-terminal extracellular loop (between the residues C131 and C172), which is required for the stability of the protein, and is probably introduced by BdbDC, a pair of competence-induced oxidoreductase proteins. By in vitro cross-linking using native cysteine residues we show that ComEC forms an oligomer. The oligomerization surface includes a transmembrane segment, TMS-G, near the cytoplasmic C-terminus of ComEC.     

The role of seladin-1/DHCR24 in cholesterol biosynthesis, APP processing and Abeta generation in vivo

The cholesterol-synthesizing enzyme seladin-1, encoded by the Dhcr24 gene, is a flavin adenine dinucleotide-dependent oxidoreductase and regulates responses to oncogenic and oxidative stimuli. It has a role in neuroprotection and is downregulated in affected neurons in Alzheimer's disease (AD). Here we show that seladin-1-deficient mouse brains had reduced levels of cholesterol and disorganized cholesterol-rich detergent-resistant membrane domains (DRMs). This was associated with inefficient plasminogen binding and plasmin activation, the displacement of beta-secretase (BACE) from DRMs to APP-containing membrane fractions, increased beta-cleavage of APP and high levels of Abeta peptides. In contrast, overexpression of seladin-1 increased both cholesterol and the recruitment of DRM components into DRM fractions, induced plasmin activation and reduced both BACE processing of APP and Abeta formation. These results establish a role of seladin-1 in the formation of DRMs and suggest that seladin-1-dependent cholesterol synthesis is involved in lowering Abeta levels. Pharmacological enhancement of seladin-1 activity may be a novel Abeta-lowering approach for the treatment of AD.     

The efficient export of NADP-containing glucose-fructose oxidoreductase to the periplasm of Zymomonas mobilis depends both on an intact twin-arginine motif in the signal peptide and on the generation of a structural export signal induced by cofactor binding.

The periplasmic, NADP-containing glucose-fructose oxidoreductase of the gram-negative bacterium Zymomonas mobilis belongs to a class of redox cofactor-dependent enzymes which are exported with the aid of a signal peptide containing a so-called twin-arginine motif. In this paper we show that the replacement of one or both arginine residues results in drastically reduced translocation of glucose-fructose oxidoreductase to the periplasm, showing that this motif is essential. Mutant proteins which, in contrast to wild-type glucose-fructose oxidoreductase, bind NADP in a looser and dissociable manner, were severely affected in the kinetics of plasma membrane translocation. These results strongly suggest that the translocation of glucose-fructose oxidoreductase into the periplasm uses a Sec-independent apparatus which recognizes, as an additional signal, a conformational change in the structure of the protein, most likely triggered by cofactor binding. Furthermore, these results suggest that glucose-fructose oxidoreductase is exported in a folded form. A glucose-fructose oxidoreductase:beta-galactosidase fusion protein is not lethal to Z. mobilis cells and leads to the accumulation of the cytosolic preform of wild-type glucose-fructose oxidoreductase expressed in trans but not of a typical Sec-substrate (OmpA), indicating that the glucose-fructose oxidoreductase translocation apparatus can be blocked without interfering with the export of essential proteins via the Sec pathway.     

Energy complexes are apparently associated with the switch-motor complex of bacterial flagella

Recently, the switch-motor complex of bacterial flagella was found to be associated with a number of non-flagellar proteins, which, in spite of not being known as belonging to the chemotaxis system, affect the function of the flagella. The observation that one of these proteins, fumarate reductase, is essentially involved in electron transport under anaerobic conditions raised the question of whether other energy-linked enzymes are associated with the switch-motor complex as well. Here, we identified two additional such enzymes in Escherichia coli. Employing fluorescence resonance energy transfer in vivo and pull-down assays invitro, we provided evidence for the interaction of F(0)F(1) ATP synthase via its β subunit with the flagellar switch protein FliG and for the interaction of NADH-ubiquinone oxidoreductase with FliG, FliM, and possibly FliN. Furthermore, we measured higher rates of ATP synthesis, ATP hydrolysis, and electron transport from NADH to oxygen in membrane areas adjacent to the flagellar motor than in other membrane areas. All these observations suggest the association of energy complexes with the flagellar switch-motor complex. Finding that deletion of the β subunit in vivo affected the direction of flagellar rotation and switching frequency further implied that the interaction of F(0)F(1) ATP synthase with FliG is important for the function of the switch of bacterial flagella.     

The ferredoxin-NADP+ oxidoreductase-binding protein is not the 17-kDa component of the cytochrome b/f complex

The complex between ferredoxin-NADP+ oxidoreductase and its proposed membrane-binding protein (Vallejos, R. H., Ceccarelli, E., and Chan, R. (1984) J. Biol. Chem. 259, 8048-8051) was isolated from spinach thylakoids and compared with isolated cytochrome b/f complex containing associated ferredoxin NADP+ oxidoreductase (Clark, R. D., and Hind, G. (1983) J. Biol. Chem. 258, 10348-10354). There was no immunological cross-reactivity between the 17.5-kDa binding protein and an antiserum raised against the 17-kDa polypeptide of the cytochrome complex. Association of ferredoxin-NADP+ oxidoreductase with the binding protein or with the thylakoid membrane gave an allotopic shift in the pH profile of diaphorase activity, as compared to the free enzyme. This effect was not seen in enzyme associated with the cytochrome b/f complex. Identification of the 17.5-kDa binding protein as the 17-kDa component of the cytochrome b/f complex is ruled out by these results.     

Functional sulfurtransferase is associated with mitochondrial complex I from Yarrowia lipolytica, but is not required for assembly of its iron-sulfur clusters

Here, we report that in the obligate aerobic yeast Yarrowia lipolytica, a protein exhibiting rhodanese (thiosulfate:cyanide sulfurtransferase) activity is associated with proton pumping NADH:ubiquinone oxidoreductase (complex I). Complex I is a key enzyme of the mitochondrial respiratory chain that contains eight iron-sulfur clusters. From a rhodanese deletion strain, we purified functional complex I that lacked the additional protein but was fully assembled and displayed no functional defects or changes in EPR signature. In contrast to previous suggestions, this indicated that the sulfurtransferase associated with Y. lipolytica complex I is not required for assembly of its iron-sulfur clusters.     

Thermodynamic properties of oxidoreductase, transferase, hydrolase, and ligase reactions

Transferases formally couple together two oxidoreductase reactions or two hydrolase reactions. Therefore the thermodynamic properties of transferase reactions can be calculated from differences between thermodynamic properties of two oxidoreductase or two hydrolase reactions. Ligases couple together two hydrolase reactions, and so their thermodynamic properties can be calculated from differences between two hydrolase reactions. These relationships are demonstrated by calculating standard transformed Gibbs energies of reaction and the changes in binding of hydrogen ions at pHs 5-9 of a number of oxidoreductase, transferase, hydrolase, and ligase reactions by use of the data base BasicBiochemData2 and its recent extensions. Coupling is not restricted to two reactions, and an example is given of the coupling of three reactions.