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We investigated H(+) and Na(+) transport by complex I from Escherichia coli devoid of the NuoL subunit, which is probably part of the ion translocating machinery. We observed that complex I devoid of the NuoL subunit still translocates H(+), although to a smaller extension than the complete version of complex I, but does not transport Na(+). Our results unequivocally reinforce the observation that E. coli complex I transports Na(+) in the opposite direction to that of the H+ and show that NuoL subunit is involved in the translocation of both ions by complex I.

Mammalian complex I is composed of fourteen highly conserved core subunits and additional thirty subunits acquired in the course of evolution. At present, the function of the majority of these supernumerary subunits is poorly understood. In this work, we have studied NDUFA3, NDUFA5 and NDUFA12 supernumerary subunits to gain insight into their role in CI activity and biogenesis. Using human cell lines in which the expression of these subunits was knocked down with miRNAs, we showed that they are necessary for the formation of a functional holoenzyme. Analysis of the assembly intermediates in mitochondria depleted for these subunits further suggested that they are required for assembly and/or stability of the electron transferring Q module in the peripheral arm of the CI., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Development of hepatocellular carcinoma (HCC) is accompanied by a continuous increase in reactive oxygen species (ROS) levels. To investigate the primary source of ROS in liver cells, we used tumor necrosis factor-alpha (TNF-α) as stimulus. Applying inhibitors against the respiratory chain complexes, we identified mitochondria as primary source of ROS production. TNF-α altered mitochondrial integrity by mimicking a mild uncoupling effect in liver cells, as indicated by a 40% reduction in membrane potential and ATP depletion (35%). TNF-α-induced ROS production activated NF-κB 3.5-fold and subsequently enhanced migration up to 12.7-fold. This study identifies complex I and complex III of the mitochondrial respiratory chain as point of release of ROS upon TNF-α stimulation of liver cells, which enhances cell migration by activating NF-κB signalling., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Although type-1 NAD(P)H dehydrogenase (NDH) complex subunit constituents and physiological functions have been reported in plants and cyanobacteria, the biochemical properties of this enzyme are not clear. We used chromatographic isolation to purify and characterize a NADPH-active NDH from the cyanobacterium Thermosynechococcus elongatus. Ferredoxin (Fd) and ferredoxin-NADP(+) oxidoreductase (FNR) were co-eluted with NDH, implying the electron donation from NADPH to NDH via the interaction with FNR. We investigated the enzymatic properties of the complex. Furthermore, the activity is competitively inhibited by rotenone, suggesting that it possesses a quinone binding site, similar to mitochondria complex I., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Pentatricopeptide repeat (PPR) proteins have been identified as site-specific factors for RNA editing in plant organelles. These proteins recognize cis-elements near the editing site. It is unclear how contiguous sites are addressed, and whether one or two factors are required. We here show the PPR MEF25 to be essential for RNA editing at the nad1-308 site in Arabidopsis mitochondria. Another editing site just one nucleotide upstream, nad1-307, is edited normally in mef25 mutant lines. This finding shows that two independent factors recognizing similar cis-elements are involved at these contiguous sites without competing with each other in vivo., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The NADH:ubiquinone oxidoreductase couples the electron transfer from NADH to ubiquinone with the translocation of protons across the membrane. It contains a 110Šlong helix running parallel to the membrane part of the complex. Deletion of the helix resulted in a reduced H(+)/e(-) stoichiometry indicating its direct involvement in proton translocation. Here, we show that the mutation of the conserved amino acid D563(L), which is part of the horizontal helix of the Escherichia coli complex I, leads to a reduced H(+)/e(-) stoichiometry. It is discussed that this residue is involved in transferring protons to the membranous proton translocation site., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

In mammals, complex I (NADH-ubiquinone oxidoreductase) of the mitochondrial respiratory chain has 31 supernumerary subunits in addition to the 14 conserved from prokaryotes to humans. Multiplicity of structural protein components, as well as of biogenesis factors, makes complex I a sensible pace-maker of mitochondrial respiration. The work reviewed here shows that the cAMP/PKA pathway regulates the biogenesis, assembly and catalytic activity of complex I and mitochondrial oxygen superoxide production. The structural, functional and regulatory complexity of complex I, renders it particularly vulnerable to genetic and sporadic pathological factors. Complex I dysfunction has, indeed, been found, to be associated with several human diseases. Knowledge of the pathogenetic mechanisms of these diseases can help to develop new therapeutic strategies., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Complex I (NADH:ubiquinone oxidoreductase) is the first and largest protein complex of the oxidative phosphorylation. Crystal structures have elucidated the positions of most subunits of bacterial evolutionary origin in the complex, but the positions of the eukaryotic subunits are unknown. Based on the analysis of sequence conservation we propose intra-molecular disulfide bridges and the inter-membrane space localization of three Cx(9)C-containing subunits in human: NDUFS5, NDUFB7 and NDUFA8. We experimentally confirm the localization of the latter two, while our data are consistent with disulfide bridges in NDUFA8. We propose these subunits stabilize the membrane domain of complex I., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

There is growing evidence of mitochondrial membrane raft-like microdomains that are involved in the apoptotic pathway. The aim of this study was to investigate the effect of methyl-beta-cyclodextrin (MβCD), being a well-known lipid microdomain disrupting agent and cholesterol chelator, on the structure and bioenergetics of rat liver mitochondria (RLM). We observed that MβCD decreases the function of RLM, induces changes in the mitochondrial configuration state and decreases the calcium chloride-induced swelling. These data suggest that disruption of mitochondrial raft-like microdomains by cholesterol efflux on one hand impairs mitochondrial bioenergetics, but on the other hand it protects the mitochondria from swelling., (Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Recently, Sazanov's group reported the X-ray structure of whole complex I [Nature, 465, 441 (2010)], which presented a strong clue for a "piston-like" structure as a key element in an "indirect" proton pump. We have studied the NuoL subunit which has a high sequence similarity to Na(+)/H(+) antiporters, as do the NuoM and N subunits. We constructed 27 site-directed NuoL mutants. Our data suggest that the H(+)/e(-) stoichiometry seems to have decreased from (4H(+)/2e(-)) in the wild-type to approximately (3H(+)/2e(-)) in NuoL mutants. We propose a revised hypothesis that each of the "direct" and the "indirect" proton pumps transports 2H(+) per 2e(-)., (Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Identification of pre-B-cell colony-enhancing factor (PBEF) interacting partners may reveal new molecular mechanisms of PBEF in the pathogenesis of acute lung injury (ALI). The interactions between PBEF and NADH dehydrogenase subunit 1(ND1), ferritin light chain and interferon induced transmembrane 3 (IFITM3) in human pulmonary vascular endothelial cells were identified and validated. ND1, ferritin and IFITM3 are involved in oxidative stress and inflammation. Overexpression of PBEF increased its interactions and intracellular oxidative stress, which can be attenuated by rotenone. The interaction modeling between PBEF and ND1 is consistent with the corresponding experimental finding. These interactions may underlie a novel role of PBEF in the pathogenesis of ALI.

Type II NADH dehydrogenases (NDH-2) are monomeric flavoenzymes catalyzing electron transfer from NADH to quinones. While most NDH-2 preferentially oxidize NADH, some of these enzymes have been reported to efficiently oxidize NADPH. With the aim to modify the NADPH vs NADH specificity of the relatively NADH specific Agrobacterium tumefaciens NDH-2, two conserved residues (E and A) of the substrate binding domain were, respectively, mutated to Q and S. We show that when E was replaced by Q at position 203 the enzyme was able to oxidize NADPH as efficiently as NADH. Growth on a minimal medium of an Escherichia coli double mutant lacking both NDH-1 and NDH-2 was restored more efficiently when mutated proteins able to oxidize NADPH were expressed. The biotechnological interest of expressing such modified enzymes in photosynthetic organisms is discussed.

We have studied the production of reactive oxygen species (ROS) in transmitochondrial cells, harboring homoplasmic levels of the T14487C mtDNA mutation in the ND6 gene of mitochondrial DNA (mtDNA). Previous work has shown that this mutation causes complex I deficiency. Here, we show that this mutation causes an overproduction of ROS leading to an increase in the oxidation of lipids and mtDNA without modification of antioxidant enzyme activities. We suggest that mutations in mtDNA affecting complex I activity may result in oxidative cellular damage, and reinforce the possible role of ROS-mediated mechanisms participating in some mtDNA-related disorders.

Cholera still remains an important global predicament especially in India and other developing countries. Vibrio cholerae, the etiologic agent of cholera, colonizes the small intestine and produces an enterotoxin that is largely responsible for the watery diarrheal symptoms of the disease. Using RNA arbitrarily primed PCR, ND5 a mitochondria encoded subunit of complex I of the mitochondrial respiratory chain was found to be upregulated in the human intestinal epithelial cell line Int407 following exposure to V. cholerae. The upregulation of ND5 was not observed when Int407 was infected with Escherichia coli strains. Incubation with heat-killed V. cholerae or cholera toxin or culture supernatant also showed no such upregulation indicating the involvement of live bacteria in the process. Infection of the monolayer with aflagellate non-motile mutant of V. cholerae O395 showed a very significant (59-fold) downregulation of ND5. In contrast, a remarkable upregulation of ND5 expression (200-fold) was observed in a hyperadherent icmF insertion mutant with reduced motility. V. cholerae cheY4 null mutant defective in adherence and motility also resulted in significantly reduced levels of ND5 expression while mutant with the cheY4 gene duplicated showing increased adherence and motility resulted in increased expression of ND5. These results clearly indicate that both motility and adherence to intestinal epithelial cells are possible triggering factors contributing to ND5 mRNA expression by V. cholerae. Interestingly infection with insertion mutant in the gene coding for ToxR, the master regulator of virulence in V. cholerae resulted in significant downregulation of ND5 expression. However, infection with ctxA or toxT insertion mutants did not show any significant changes in ND5 expression compared to wild-type. Almost no expression of ND5 was observed in case of mutation in the gene coding for OmpU, a ToxR activated protein. Thus, infection of Int407 with virulence mutant strains of V. cholerae revealed that the ND5 expression is modulated by the virulence of V. cholerae in a ToxT independent manner. Although no difference in the mitochondrial copy number could be detected between infected and uninfected cells, the modulation of the expression of other mitochondrial genes were also observed. Incidentally, upon V. cholerae infection, complex I activity was found to increase about 3-folds after 6 h. This is the first report of alteration in mitochondrial gene expression upon infection of a non-invasive enteric bacterium like V. cholerae showing its modulation with adherence, motility and virulence of the organism.

Properties of Klebsiella pneumoniae respiratory chain enzymes catalyzing NADH oxidation have been studied. Using constructed K. pneumoniae mutant strains, it was shown that three enzymes belonging to different families of NADH:quinone oxidoreductases operate in this bacterium. The NDH-2-type enzyme is not coupled with energy conservation, the NDH-1-type enzyme is a primary proton pump, and the NQR-type enzyme is homologous to the sodium-motive NADH dehydrogenase of Vibrio and is shown to be a primary Na(+) pump. It is concluded that the NQR-type enzyme, not the NDH-1-type enzyme, catalyzes sodium-dependent NADH oxidation in K. pneumoniae.

We studied the effect of ageing on the mRNA levels of mitochondria-encoded polypeptides in human platelets. We used quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) to investigate the expression of selected cytochrome c oxidase (COX) genes (subunits I and III) and Complex I genes (subunits reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase (ND)1 and (ND)5 in platelets from young and aged healthy subjects. Northern blot analysis confirmed the PCR results. COX I expression is higher than that of COX III in both young and aged platelets. A significant increase of transcripts for Complex I was found during ageing. On the contrary, the mRNA levels of the two COX subunits did not significantly vary during ageing.

Challenge of Rhodobacter capsulatus cells with the superoxide propagator methyl viologen resulted in the induction of a diaphorase activity identified as a member of the ferredoxin (flavodoxin)-(reduced) nicotinamide adenine dinucleotide phosphate (NADP(H)) reductase (FPR) family by N-terminal sequencing. The gene coding for Rhodobacter FPR was cloned and expressed in Escherichia coli. Both native and recombinant forms of the enzyme were purified to homogeneity rendering monomeric products of approximately 30 kDa with essentially the same spectroscopic and kinetic properties. They were able to bind and reduce Rhodobacter flavodoxin (NifF) and to mediate typical FPR activities such as the NADPH-driven diaphorase and cytochrome c reductase.

The thermoacidophilic archaeon Acidianus ambivalens contains a monomeric 47 kDa type-II NADH dehydrogenase (NDH), which contains a covalently bound flavin. In this work, by a combination of several methods, namely (31)P-nuclear magnetic resonance and fluorescence spectroscopies, it is proven that this enzyme contains covalent FMN, a novelty among this family of enzymes, which were so far thought to mainly have the flavin dinucleotide form. Discrimination between several possible covalent flavin linkages was achieved by spectral and fluorescence experiments, which identified an 8alpha-N(1)-histidylflavin-type of linkage. Analysis of the gene-deduced amino acid sequence of type-II NDH showed no transmembranar helices and allowed the definition of putative dinucleotide and quinone binding motifs. Further, it is suggested that membrane anchoring can be achieved via amphipatic helices.

Cold stress effects on the expression of genes for respiratory chain enzymes were investigated in potato (Solanum tuberosum L., cv. Desiree) leaves. The nda1 and ndb1 genes, homologues to genes encoding the non-proton-pumping respiratory chain NADH dehydrogenases of Escherichia coli and yeast, were compared to genes encoding catalytic subunits of the proton-pumping NADH dehydrogenase (complex I). Using a real-time PCR system, we demonstrate a specific and gradual decrease of the NDA1 transcript after exposing the plants to 5 degrees C. After 6 days of cold treatment the NDA1 transcript abundance is 10% of the original level. This decrease is accompanied by specific decreases of immunodetected NDA protein and internal rotenone-insensitive NADH oxidation in mitochondria isolated from cold-treated plants. The alternative oxidase is not cold-induced neither at the protein nor at the activity level. The results are discussed in relation to the recent finding that the nda1 gene expression is completely light-dependent.

The NADH:ubiquinone reductase (NDH-2) of Escherichia coli was expressed as a His-tagged protein, extracted from the membrane fraction using detergent and purified by chromatography. The His-tagged NDH-2 was highly active and catalyzed NADH oxidation by ubiquinone-1 at rates over two orders of magnitude higher than previously reported. The purified, His-tagged NDH-2, like native NDH-2, did not oxidize deamino-NADH. Steady-state kinetics were used to analyze the enzyme's activity in the presence of different electron acceptors. High V(max) and low K(m) values were only found for hydrophobic ubiquinone analogues, particularly ubiquinone-2. These findings strongly support the notion that NDH-2 is a membrane bound enzyme, despite the absence of predicted transmembrane segments in its primary structure. The latter observation is in agreement with possible evolutionary relation between NDH-2 and water-soluble enzymes such as dihydrolipoamide dehydrogenase. There is currently no clear indication of how NDH-2 binds to biological membranes.

Subunit 6 of the yeast cytochrome bc1 complex contains a 25 amino acid presequence that is not present in the mature form of the protein in the bc1 complex. The presequence of subunit 6 is atypical of presequences responsible for targeting proteins to mitochondria. Whereas mitochondrial targeting sequences rarely contain acidic residues and typically contain basic residues that can potentially form an amphiphilic structure, the presequence of subunit 6 contains only one basic amino acid and is enriched in acidic amino acids. If the 25 amino acid presequence is deleted, subunit 6 is imported into mitochondria and assembled into the cytochrome bc1 complex and the activity of the bc1 complex is identical to that from a wild-type yeast strain. However, if the C-terminal 45 amino acids are truncated from the protein, subunit 6 is not present in the mitochondria and the activity of the bc1 complex is diminished by half, identical to that of the bc1 complex from a yeast strain in which the QCR6 gene is deleted. These results indicate that the presequence of subunit 6 is not required for targeting to mitochondria or assembly of the subunit into the bc1 complex and that information necessary for targeting and import into mitochondria may be present in the C-terminus of the protein.

An NADPH dependent cytochrome c reductase has been purified from resting bovine neutrophil membranes. A high degree of purification, approaching homogeneity, is indicated by the presence of a single 75 kDa protein band on silver stained SDS-PAGE (10%). The purified protein catalyzes as well an NADPH dependent reduction of iodonitrotetrazolium violet (INT). Limited papain digestion of the purified preparation produces a 65 kDa product which retains both enzymatic activities. In a similar fashion papain digestion of the plasma membrane bound protein generates a fully active soluble NADPH dependent INT and cytochrome c reductase preparation (65 kDa). Proteolytic cleavage would appear to occur at a protein-membrane anchor remote from the proteins catalytic site. The cytochrome c reductase acts independently of the O2-generating cytochrome b558, a leukocyte plasma membrane protein which also catalyzes an NADPH dependent INT reduction.

Genes that encode mitochondrial homologues to the bacterial enzymes of fatty acid synthesis were found in various eukaryotic species. Inactivation of these genes leads to a disturbed mitochondrial respiration and an increase in mitochondrial lysophospholipids. We postulate that there is a mitochondrial biosynthetic system providing fatty acids for phospholipid repair. The mitochondrial acyl carrier protein may also play another role, supporting the formation of the respiratory NADH:ubiquinone oxidoreductase.

The H(+)-translocating NADH:ubiquinone oxidoreductase (NDH1) is probably an obligatory enzyme in Paracoccus denitrificans and disruption of its genes may be lethal to this organism. In order to overcome this problem and delete the nqo8 and nqo9 genes of NDH1, it was necessary to render the enzyme non-essential. This was achieved by constructing a deletion plasmid in which most of the coding regions of nqo8 and nqo9 were replaced by the ndh gene of Escherichia coli that encodes an alternative NADH:ubiquinone oxidoreductase (NDH2), and a kanamycin resistance gene. Subsequent homologous recombination gave rise to a mutant the membranes of which catalyzed rotenone-insensitive NADH oxidation, but which did not oxidize deamino-NADH. Hence, this mutant expressed active and membrane-bound NDH2, and lacked NDH1 activity.

Using non-denaturing gel electrophoresis and staining with nitro-blue tetrazolium, we reveal the presence of two NAD(P)H oxidoreductase activity bands within thylakoids membranes of Solanum tuberosum L. Second dimension SDS-PAGE and Western analysis show that one of the activity bands contains several polypeptides, two of them being recognized by antibodies directed against peptides corresponding to conserved domains of chloroplastic genes products NDH B and NDH J (at 32 and 18 kDa, respectively). Both activity bands also contain a polypeptide (around 36 kDa) recognized by an antibody directed against ferredoxin-NADP(+)-reductase (FNR). We conclude from these results that both chloroplastic ndh B and ndh J gene products are components of a thylakoid NAD(P)H dehydrogenase complex. The association with FNR is suggested to allow the complex to use NADPH instead of NADH as a preferential substrate.

Exogenous NADPH oxidation by purified mitochondria from both potato tuber and Arum maculatum spadix was completely and irreversibly inhibited by sub-micromolar diphenyleneiodonium (DPI), while exogenous NADH oxidation was inhibited to only a small degree. Addition of DPI caused the collapse of the membrane potential generated by NADPH oxidation, while the potential generated by NADH was unaffected. We conclude that there are two distinct enzymes on the outer surface of the inner membrane of plant mitochondria, one specific for NADH, the other relatively specific for NADPH, with both enzymes linked to the electron transport chain.

It was found that the activities of prooxidant enzymes (NAD(P)H oxidases and NAD(P)H:cytochrome c reductases) in bovine leukemia virus-transformed calf and lamb embryo kidney fibroblasts (lines Mi-18 and FLK) were by 1.25-18 times higher when compared to corresponding nontransformed calf cells. The activity of DT-diaphorase was also increased by about one order of magnitude in transformed cells. The activities of antioxidant enzymes were almost unchanged (superoxide dismutase), decreased by 13% or 53% (catalase) or increased by 25% or 90% (glutathione reductase) in Mi-18 or FLK cells, respectively. These changes of enzyme activity increased the toxicity of simple redox-cycling quinones (duroquinone, naphthazarin) towards transformed cells, but did not affect the toxicity of daunorubicin. The latter was most probably related to the inhibition of plasma membrane NADH dehydrogenase.

The saturation kinetics of NADH and succinate oxidation for Coenzyme Q (CoQ) has been re-investigated in pentane-extracted lyophilized beef heart mitochondria reconstituted with exogenous CoQ10. The apparent 'Km' for CoQ10 was one order of magnitude lower in succinate cytochrome c reductase than in NADH cytochrome c reductase. The Km value in NADH oxidation approaches the natural CoQ content of beef heart mitochondria, whereas that in succinate oxidation is close to the content of respiratory chain enzymes.

We report the electron transfer properties of the NADH:ubiquinone oxidoreductase complex of the respiratory chain (Complex I) in mitochondria of cells derived from LHON patients with two different mutations in mitochondrial DNA (mtDNA). The mutations occur in the mtDNA genes coding for the ND1 and ND4 subunits of Complex I. The ND1/3460 mutation exhibits 80% reduction in rotenone-sensitive and ubiquinone-dependent electron transfer activity, whereas the proximal NADH dehydrogenase activity of the Complex is unaffected. This is in accordance with the proposal that the ND1 subunit interacts with rotenone and ubiquinone. In contrast, the ND4/11778 mutation had no effect on electron transfer activity of the Complex in inner mitochondrial membrane preparations; also Km for NADH and NADH dehydrogenase activity were unaffected. However, in isolated mitochondria with the ND4 mutation, the rate of oxidation of NAD-linked substrates, but not of succinate, was significantly decreased. This suggests that the ND4 subunit might be involved in specific aggregation of NADH-dependent dehydrogenases and Complex I, which may result in fast ('solid state') electron transfer from the former to the latter.

The H+/e- stoichiometry of protonmotive cytochrome c oxidase, isolated from bovine heart mitochondria and reconstituted in liposomes, has been determined by making use of direct spectrophotometric measurements of the initial rates of e- flow and H+ translocation. It is shown that the ----H+/e- ratio for redox-linked proton ejection by the oxidase varies from around 0 to a maximum of 1 as a function of the rate of overall electron flow in the complex.

A study is presented of the ----H+/e- stoichiometry for H+ pumping by the cytochrome chain in isolated rat liver mitochondria under level-flow and steady-state conditions. It is shown that the ----H+/e- stoichiometry for the cytochrome chain varies under the influence of the flow rate and transmembrane delta microH+. The rate-dependence is shown to be associated with cytochrome c oxidase, whose ----H+/e- ratio varies from 0 to 1, whilst the ----H+/e- ratio for the span covered by cytochrome c reductase is invariably 2.

Cytochrome c reductase is inhibited by p-chlorophenyl-methoxybenzyl-ketoxime (CPMB-oxime). CPMB-oxime induces a red-shift of the reduced spectrum of cytochrome b. The inhibitor blocks the oxidation of ubihydroquinone at the QP center of this enzyme in a non-competitive way. The binding stoichiometry equals one inhibitor molecule per Qp center. The apparent Kd in a red-shift assay was 6.9 +/- 0.6 microM. All binding characteristics analysed in this study were very similar to those of the E-beta-methoxyacrylate inhibitors, although the chemical structure is different from these inhibitors. This result is interpreted as a support for the inhibitory mechanism based on the model of a 'catalytic switch' proposed recently for the E-beta-methoxyacrylate inhibitors (MOA-inhibitors (Brandt and von Jagow, Eur. J. Biochem.

Electron transfer through the ubiquinol:cytochrome c1-segment of liver mitochondria isolated from hibernating ground squirrels Citellus undulatus is repressed by 70-80% as compared to mitochondria from the active animals. The inhibition site is likely to be localized between ubiquinone and the cytochrome bc1 complex. Partial release of the inhibition can be observed upon swelling of the isolated mitochondria in a hypoosmotic medium, the effect being prevented by phospholipase A2 inhibitors. Possible role of phospholipase A2 in regulation of ubiquinol oxidation by complex bc1 is discussed.

Cell-free extracts of methanol-grown Nocardia sp. 239 only show significant dye-linked methanol-oxidizing activity when NAD+ is added to the assay mixture. This activity resides in a multienzyme complex which could be resolved into 3 components, namely the methanol dehydrogenase, NAD-dependent aldehyde dehydrogenase and NADH dehydrogenase. In its dissociated form, the methanol dehydrogenase no longer shows dye reduction and although rises in the absorbance values around 340 nm are seen on addition of methanol plus NAD+ to the enzyme, this is not due to NADH production. However, dye reduction (NAD dependent) could be restored on incubating methanol dehydrogenase with the corresponding NADH dehydrogenase, obtained from the enzyme complex. It is concluded that this novel methanol dehydrogenase transfers the reducing equivalents, derived from methanol, directly to its associated NADH dehydrogenase via a mechanism in which NAD+ and PQQ are involved.

The delta 9-desaturase system in liver microsome from rats treated chronically with ethanol was studied. Stearoyl-CoA desaturase activity decreased by 80% and palmitoyl-CoA desaturase activity was not detectable in microsomes from ethanol-fed rats, while activities of electron transport components such as NADH-cytochrome c and NADH-ferricyanide reductases remained unchanged. However, chronic ethanol administration resulted in an adaptive induction of the activity of NADPH-cytochrome c reductase and the contents of cytochrome b5 and P-450. The activity of the terminal component (cyanide-sensitive factor; CSF) of the desaturase system was greatly depressed by ethanol treatment. The NADH/NAD ratio in microsomes of ethanol-fed rats increased over 2-fold. These results suggest that, during chronic ethanol ingestion, decreased activities of delta 9-desaturases are due mainly to a decreased content of the terminal component of the desaturase system.

Mitochondrial NADH dehydrogenase from bovine heart was photolabelled with the inhibitor [3H]dihydrorotenone. A constituent of the hydrophobic domain of the enzyme of Mr 33,000 was the major site of labelling. The identity of this protein with the mitochondrially encoded ND-1 gene product was established by immunoblotting and immunoprecipitation with an antiserum raised to the expected C-terminal sequence of the human ND-1 gene product.

Alkalophile NADH dehydrogenase consisting of two 65-kDa subunits was changed by subtilisin into an enzyme species consisting of two 38-kDa subunits. The amino acid composition and enzyme activity per molecule of the subtilisin-treated enzyme were almost the same as those of the native enzyme, respectively. On mixing with phospholipid liposome, the conformation of the native enzyme was changed, as suggested by the changes in the type of Arrhenius plot and of CD spectrum and enzyme activity. These conformational properties of the subtilisin-treated enzyme, on the other hand, were not affected by liposome. Gel filtration of the subtilisin-treated enzyme mixed with the liposome showed no binding of the protein to liposome.

Addition of NADH, but not NAD+ or NADPH, to rat liver plasma membranes resulted in the increase of their 5'-nucleotidase activity. NADH-dependent activation of 5'-nucleotidase was significantly suppressed by atebrine, an inhibitor of NADH dehydrogenase of plasma membranes, and completely abolished by 2,4-dinitrophenol (2 X 10(-4)M) and Triton X-100 (2%). Inhibitors of electron transfer in the mitochondrial respiratory chain, rotenone and potassium cyanide, failed to affect 5'-nucleotidase activity in both the presence and absence of NADH. The data obtained give reasons to suggest a redox-dependent mechanism of 5'-nucleotidase activation in rat liver plasma membranes.