Multiplicity of monoamine oxidase: inhibition of mitochondrial monoamine oxidase activity by isopropylhydrazide of D,L-serine]

Isopropylhydrazide of D,L-serine (IHS) inhibits by 50% (at 37 degrees for 10 min) deamination of serotonin or beta-phenylethylamine by monoamine oxidases from bovine brain stem mitochondrial membranes at the 2.6 X X 10(-5) M or 9 X 10(-5) M, respectively. In order to inhibit by 50% the deamination of tyramine under the same conditions a considerably lower (2.5 X X 10(-6) M) concentration of IHS is required. Kinetic studies of inhibition of enzymatic deamination of all the three biogenic monoamines by IHS showed that the irreversible blocking of the monoamine oxidase activity is preceeded by formation of dissociating enzyme-inhibitor complexes. Values of the dissociation constants of these complexes measured (at 37 degrees) with serotonin, phenylethylamine or tyramine as substrates for estimation of the residual monoamine oxidase activity are 0.47; 0.13 or 0.023 mM, respectively. Significant differences are also found between thermodynamic and activation parameters characterizing both both steps of interaction between IHS and the monoamine oxidases of mitochondrial membranes in the experiments with serotonin, phenylethylamine or tyramine as substrates. The data obtained suggest the existence of different monoamine oxidases (or their active sites) catalyzing oxidative deamination of serotonin, phenylethylamine or tyramine in the fragments of mitochondrial membranes from bovine brain stem.     

Yeast ERV2p is the first microsomal FAD-linked sulfhydryl oxidase of the Erv1p/Alrp protein family

Saccharomyces cerevisiae Erv2p was identified previously as a distant homologue of Erv1p, an essential mitochondrial protein exhibiting sulfhydryl oxidase activity. Expression of the ERV2 (essential for respiration and vegetative growth 2) gene from a high-copy plasmid cannot substitute for the lack of ERV1, suggesting that the two proteins perform nonredundant functions. Here, we show that the deletion of the ERV2 gene or the depletion of Erv2p by regulated gene expression is not associated with any detectable growth defects. Erv2p is located in the microsomal fraction, distinguishing it from the mitochondrial Erv1p. Despite their distinct subcellular localization, the two proteins exhibit functional similarities. Both form dimers in vivo and in vitro, contain a conserved YPCXXC motif in their carboxyl-terminal part, bind flavin adenine dinucleotide (FAD) as a cofactor, and catalyze the formation of disulfide bonds in protein substrates. The catalytic activity, the ability to form dimers, and the binding of FAD are associated with the carboxyl-terminal domain of the protein. Our findings identify Erv2p as the first microsomal member of the Erv1p/Alrp protein family of FAD-linked sulfhydryl oxidases. We propose that Erv2p functions in the generation of microsomal disulfide bonds acting in parallel with Ero1p, the essential, FAD-dependent oxidase of protein disulfide isomerase.     

Acyl-CoA oxidase 1 from Arabidopsis thaliana. Structure of a key enzyme in plant lipid metabolism

The peroxisomal acyl-CoA oxidase family plays an essential role in lipid metabolism by catalyzing the conversion of acyl-CoA into trans-2-enoyl-CoA during fatty acid beta-oxidation. Here, we report the X-ray structure of the FAD-containing Arabidopsis thaliana acyl-CoA oxidase 1 (ACX1), the first three-dimensional structure of a plant acyl-CoA oxidase. Like other acyl-CoA oxidases, the enzyme is a dimer and it has a fold resembling that of mammalian acyl-CoA oxidase. A comparative analysis including mammalian acyl-CoA oxidase and the related tetrameric mitochondrial acyl-CoA dehydrogenases reveals a substrate-binding architecture that explains the observed preference for long-chained, mono-unsaturated substrates in ACX1. Two anions are found at the ACX1 dimer interface and for the first time the presence of a disulfide bridge in a peroxisomal protein has been observed. The functional differences between the peroxisomal acyl-CoA oxidases and the mitochondrial acyl-CoA dehydrogenases are attributed to structural differences in the FAD environments.     

The effect of long-term inhibition of mitochondrial protein synthesis on the oxidation capacity of mitochondria for NADH-linked substrates

Experiments are presented showing that specific inhibition of mitochondrial protein synthesis by tetracyclines decreases the activity of the NADH-dehydrogenase complex in liver mitochondria, if rats are treated for long periods with these antibiotics. The corresponding inhibition of this complex in tumor cells (Zajdela hepatoma) and tumor mitochondria (Leydig cell tumor) is even more pronounced. It is concluded that the mitochondrial genetic system is involved in the assembly of the NADH-dehydrogenase complex, most likely by coding for one or more subunits. It is argued that this information, contrary to the situation for cytochrome c oxidase, the cytochrome bc1 complex and ATPsynthase, has been missed in previous experiments employing differential inhibition of mitochondrial protein synthesis, because of the circumstance that the inhibition did not reach the level at which it became rate-limiting.     

Glutathione dependent control of protein disulfide-sulfhydryl content by subcellular fractions of hepatic tissue

The disulfide-sulfhydryl (SS/SH) ratios of subcellular fractions of rat hepatic tissue were found to vary diurnally with the ratio lowest in the early morning and highest in the early evening. These changes were found in the nuclear, microsomal and cytosol fractions. The primary reaction is the reversible formation of mixed disulfides of glutathione with proteins. This formation is controlled by the activity of thiol transferase and the level of oxidized glutathione (GSSG) as substrate. Several enzymes including mitochondrial and microsomal oxidases, glutathione reductase and peroxidase and glucose-6-phosphate dehydrogenase were found to control the levels of GSSG. An NADPH-dependent microsomal oxidase system, inhibited by GSSG, was found to produce activated oxygen which served as substrate for flutathione peroxidase. Evidence is presented for the concept that the formation of mixed disulfides of proteins with glutathione is a mechanism for maintenance of a disulfide-sulfhydryl ratio such that the integrity of particulate membranes is maintaine during oxidative and reductive stresses on the hepatic cells.     

Effects of L-malate on mitochondrial oxidoreductases in liver of aged rats

Accumulation of oxidative damage has been implicated to be a major causative factor in the decline in physiological functions that occur during the aging process. The mitochondrial respiratory chain is a powerful source of reactive oxygen species (ROS), considered as the pathogenic agent of many diseases and aging. L-malate, a tricarboxylic acid cycle intermediate, plays an important role in transporting NADH from cytosol to mitochondria for energy production. Previous studies in our laboratory reported L-malate as a free radical scavenger in aged rats. In the present study we focused on the effect of L-malate on the activities of electron transport chain in young and aged rats. We found that mitochondrial membrane potential (MMP) and the activities of succinate dehydrogenase, NADH-cytochrome c oxidoreductase and cytochrome c oxidase in liver of aged rats were significantly decreased when compared to young control rats. Supplementation of L-malate to aged rats for 30 days slightly increased MMP and improved the activities of NADH-dehydrogenase, NADH-cytochrome c oxidoreductase and cytochrome c oxidase in liver of aged rats when compared with aged control rats. In young rats, L-malate administration increased only the activity of NADH-dehydrogenase. Our result suggested that L-malate could improve the activities of electron transport chain enzymes in aged rats.     

Developmental Changes in Rat Liver Xanthine Oxidase(s) and Its Intracellular Distribution

Developmental change and subcellular distribution of xanthine oxidase in the rat liver were examined.

The specific activity of the fetal liver xanthine oxidase increased sharply to the levels of the adult liver on the day of the birth. After birth, the activity dropped rapidly and on the 14th day after birth it was about 1/4 of adult level. Then the activity was regained and around 28th day after birth it was about the same as in adult level.

In the livers from 80 days old rats, about 60% of total xanthine oxidase activity was found in soluble fraction and the rest was distributed among particulate fractions including microsomal, lysosomal, mitochondrial and nuclear fractions.

In contrast to the adult livers 80% of total xanthine oxidase activity in fetal liver was found to be in particulate fractions.

From kinetic studies of xanthine oxidases in particulate and soluble fractions it was suggested that xanthine oxidase in soluble fraction and xanthine oxidase in particulate fraction might be different in their natures of protein molecule.     

Methotrexate: studies on the cellular metabolism. I. Effect on mitochondrial oxygen uptake and oxidative phosphorylation

Effect of methotrexate (MTX) on mitochondrial oxygen uptake, oxidative phosphorylation and on the activity of several enzymes linked to respiratory chain was studied. MTX was able to inhibit state III respiration activated by ADP and to decrease the respiratory coefficient with the substrates alpha-ketoglutarate and glutamate; these effects became pronounced when mitochondria were pre-incubated with MTX for 10 min. No effect was observed on ATPase activity of undamaged or broken mitochondria; the same was true for NADH-oxidase, NADH-dehydrogenase, NADH-cytochrome c reductase, succinate oxidase, and cytochrome c oxidase activity. The effect on the steady-state of cytochrome b, as well as, the inhibitory effect on state III of respiration with NAD+-linked substrates, offers a reasonable possibility to suggesting that the inhibition site of MTX could be in a place anterior to cytochrome b region, and not linked to respiratory chain.     

Terminal oxidases in the trypanosomatid Trypanosoma cruzi

Titration of Trypanosoma cruzi respiration with cyanide, with results treated as Dixon plots, indicated the presence of several terminal oxidases. The inhibitions obtained at low cyanide concentrations (0-300 microM), taken together with cyanide effects on cytochrome aa3-deficient, dyskinetoplastic epimastigotes, supported cytochrome aa3 as T. cruzi main terminal oxidase. By increasing cyanide concentration to 1.0 mM, two alternative terminal oxidases could be detected. One of these was active in both kinetoplastic and dyskinetoplastic (cytochrome aa3-deficient) epimastigotes, and azide- and antimycin-insensitive. Complementary cytochrome studies with intact epimastigotes and mitochondrial membranes revealed the presence of cytochromes aa3, b, c558, o and possibly d, as components of the parasite electron transport system. Fractionation studies demonstrated that both o and d were bound to the mitochondrial membrane. Reduction by endogenous substrates and complex formation with cyanide supported cytochrome o as alternative terminal oxidase. EB-cultured, dyskinetoplastic epimastigotes showed the same respiration rate as the kinetoplastic cells, despite the significant decrease of cytochrome aa3, thus indicating adaptive mechanisms that determine the expression of alternative oxidases, whenever the main terminal activity is depressed.     

Sequence homology of bacterial and mitochondrial cytochrome c oxidases: Partial sequence data of cytochrome c oxidase from Paracoccus denitrificans

The aerobic electron transport chain of $\text{Paracoccus denitrificans}$ is very similar to that of mitochondria. It has therefore been suggested that this bacterium might be evolutionarily related to mitochondria. The two subunits (Mr 45.000 and 28.000) of the $\text{Paracoccus}$ cytochrome c oxidase were isolated and partially sequenced. The sequences were found to be surprisingly homologous to sequences of the subunits I and II of mitochondrial cytochrome c oxidases. The data provide a molecular basis for the symbiotic origin of mitochondria and strongly support the notion that in eucaryotic oxidases subunits I and/or II carry the redox centers, heme and copper.     

The effect of yeast dehydration on the activity of a number of enzymes of cell energetic metabolism

Summary It has been shown that dehydration markedly affects the activity of a number of enzymes connected with energy metabolism in the yeastSaccharomyces cerevisiae. Independently of the drying method used, there was found to be an inverse relationship between the activity of mitochondrial enzymes — NADH-dehydrogenase (EC 1.6.2.1), succinate dehydrogenase (EC 1.3.99.1) and cytochrome C oxidase (EC 1.9.3.1) - and the viability of yeast cells at the stationary growth phase. Dehydration led to an increase in activity only in exogenous NADH-dehydrogenase compared with activity in the initial compressed yeast. On the basis of alcohol dehydrogenase (EC 1.1.1.1) and catalase (EC 1.11.1.6) as examples, an ambivalent effect of the dehydration process on the activity of cytoplasmic enzymes has been demonstrated. The results obtained lead to the conclusion that the activity of individual electron-transport enzymes in yeastSaccharomyces cerevisiae is a sufficiently sensitive to be used as an indicator of the physiological state and to monitor a microbial biomass dehydration procedure.     

Changes in mitochondrial heterogenicity during aerobic growth of Saccharomyces cerevisiae yeasts]

Distribution of the activities of some mitochondrial enzymes after sucrose density gradient ultracentrifugation of cell homogenates of S. cerevisiae in the early and late exponential growth phases is studied. It is demonstrated that young yeast cells have a characteristic complex distribution of NADH oxidase (cyanide-sensitive), succinate:ferricyanide-oxidoreductase (or succinate:2,6-dichlorophenol indophenol-oxidoreductase), NADH:2,6-dichlorophenol indophenol-oxidoreductase and cytochrome oxidase activities in sucrose density gradient; the distribution patterns of these activities are different. All the above activities are detected in a single relatively narrow band in mature yeast cells. Similar results are obtained in the experiments with glucose or galactose as a carbon source in the yeast growth media. The Arrhenius plots for NADH oxidase (as well as for succinate:2,6-dichlorophenol indophenol-oxidoreductase) activity do not differ in the case of "light" and "heavy" mitochondrial structures characteristic of yeast cells in the early exponential growth phase. Nevertheless, "light" and "heavy" mitochondrial structures differ with respect of the arrangement of certain respiratory chain components in their membranes NADH-dehydrogenase and cytochrome oxidase). This conclusion is drawn from the results obtained in the study of the interaction of the two types of structures with Fe(CN)6(3-), a non-penetrating ion and the antiserum to yeast mitochondria.     

Do mammalian amine oxidases and the mitochondrial polyamine transporter have similar protein structures?

Polyamine transport across the mitochondria membrane occurs by a specific, common uniporter system and appears controlled by electrostatic interactions as for polyamine oxidative deamination by bovine serum and mitochondrial matrix amine oxidases was found. In fact in all the cases, while the catalytic constants or the maximum uptake rate values show little changes with the number of the positive charges of the substrates, Michaelis–Menten constant values demonstrate exponential dependence, confirming that electrostatic forces control the docking of the substrate into the enzyme active site or polyamine channel. By the treatment of the kinetic data in terms of Gibbs equation or Eyring theory, the contribution of each positive charge of the polyamine to the Gibbs energy values for the oxidative deamination of polyamines by two mammalian amine oxidase and for polyamine transport, are obtained. These values were comparable and in good accordance with those reported in literature. Previous studies demonstrated that two negative functional groups in the active site of bovine serum and mitochondrial matrix amine oxidases interact electrostatically with three positive charges of the polyamines in the formation of the enzyme–substrate complex. Remembering the structure–function relationship of proteins, our results suggest analogous interactions in the polyamine transporter and, as a consequence, a partial structural similitude between two proteins. It follows that the primary sequences of the amino oxidases and the mitochondrial transport may, in part, be conserved.     

Inhibitory analysis of the functions of the oxidative metabolic systems of tumor cells in tissue culture

As has been previously reported an increased intensity of light-induced green fluorescence is observed for some tumor cells. The present paper deals with the cause of this phenomenon, employing for this hepatoma cells of line HTC acted upon with 2,4-DNP, amytal and malonate. It has been shown that the light-induced increase in green fluorescence in cells is due to the oxidation of NADH-dehydrogenase, a mitochondrial flavine-containing enzyme, occurring at the time of fluorescence induction. The increased intensity of green fluorescence of flavoproteins in tumor cells is associated with an infringement in oxidation of NAD-dependent substrates in these, and with the activation of the reverse electron transport in the oxidative chain. The exciding light activates NADH-dehydrogenase and accelerates the translocation of reduced equivalents from this enzyme, which results in its oxidation, and thus--in the observed effect of increased intensity of green fluorescence.     

Feprazone: an inducer of the P450 II B family of proteins in the rat

The ability of feprazone to induce the hepatic microsomal mixed-function oxidases was investigated in the rat, with emphasis being placed on the nature of the cytochrome P-450 family induced. Treatment with feprazone enhanced the p-hydroxylation of aniline and the dealkylations of benzphetamine and pentoxyresorufin but had no effect on the O-deethylation of ethoxyresorufin. The same treatment had no major effect on total cytochrome P-450 levels but increased the spectral interaction of metyrapone with reduced cytochrome P-450. Immunoblots employing monospecific polyclonal antibodies revealed that feprazone induces the apoprotein levels of the P450 II B, but not of the P450 I, family. It is concluded that feprazone is an inducer of the rat hepatic mixed-function oxidase system showing selectivity toward the P450 II B family.     

Cytokinin oxidase: Biochemical features and physiological significance

The catabolism of cytokinin in plant tissues appears to be due, in large part, to the activity of a specific enzyme, cytokinin oxidase. This enzyme catalyses the oxidation of cytokinin substrates bearing unsaturated isoprenoid side chains, using molecular oxygen as the oxidant. In general, substrate specificity is highly conserved and cytokinin substrates bearing saturated or cyclic side chains do not serve as substrates for most cytokinin oxidases tested to date. Despite variation in molecular properties of the enzyme from a number of higher plants, oxygen is always required for the reaction. Cytokinin oxidases from several sources have been shown to be glycosylated. Cytokinin oxidase activity appears to be universally inhibited by cytokinin-active urea derivatives. Auxin has been reported to act as an allosteric regulator which increases activity of the enzyme.
Cytokinin oxidase activity is subject to tight regulation. Levels of the enzyme are controlled by a mechanism sensitive to cytokinin supply. The up-regulation of cytokinin oxidase expression in response to exogenous application of cytokinin suggests that the metabolic fate of exogenously applied cytokinins may not accurately mimic that of the endogenous compounds.
Cytokinin oxidase is believed to be a copper-containing amine oxidase (EC 1.4.3.6). Considerable evidence strongly supports a common mechanism for amine oxidases. It is possible that advances in understanding of other amine oxidases could be extrapolated to increase our understanding of cytokinin oxidase at the molecular level. This is discussed with reference to what is currently known about the catalytic mechanism of the enzyme. The possibility of pyrroloquinoline quinone, or a closely related compound, as a redox cofactor of cytokinin oxidase is considered, as are the implications of the glycosylated nature of the enzyme for its regulation and compartmentalisation within the cell.     

Multiple amine oxidases in cucumber seedlings

Cell-free extracts of cucumber (Cucumis sativus L. cv. National Pickling) seedlings were found to have amine oxidase activity when assayed with tryptamine as a substrate. Studies of the effect of lowered pH on the extract indicated that this activity was heterogeneous, and three amine oxidases could be separated by ion exchange chromatography. The partially purified enzymes were tested for their activities with several substrates and for their sensitivities to various amine oxidase inhibitors. One of the enzymes may be a monoamine oxidase, although it is inhibited by some diamine oxidase inhibitors. The other two enzymes have properties more characteristic of the diamine oxidases. The possible relationship of the amine oxidases to indoleacetic acid biosynthesis in cucumber seedlings is discussed.     

Differential Carbon Monoxide Sensitivity of Cytochrome-Oxidase in the Leaves of Tall and Dwarf Wheat Cultivars

Differential response in the leaves of tall and dwarf wheat to CO, an inhibitor of cytochrome oxidase and to SHAM, an inhibitor of alternative oxidase appears to be correlated with presence of Rht dwarfing genes. This was detected by in vivo nitrate reductase assay after CO treatment and direct O₂ uptake in presence of SHAM. Pretreatment of the leaves with Triton X-100 at a concentration which specifically inhibits the accessibility of exogenous NAD(P)H to alternative oxidase, Significantly enhanced the CO response as assessed by in vivo NR assay. This supports the hypothesis that the competition for NADH between NR and mitochondrial respiration is regulated by NADH-dehydrogenase located on the outer surface of inner mitochondrial membrane.     

Horizontal Gene Transfer Involved in the Convergent Evolution of the Plasmid-Encoded Enantioselective 6-Hydroxynicotine Oxidases

The D- and L-specific nicotine oxidases are flavoproteins involved in the oxidative degradation of nicotine by the Gram-positive soil bacterium Arthrobacter nicotinovorans. Their structural genes are located on a 160-kbp plasmid together with those of other nicotine-degrading enzymes. They are structurally unrelated at the DNA as well as at the protein level. Each of these oxidases possesses a high degree of substrate specificity; their catalytic stereoselectivity is absolute, although they are able to bind both enantiomeric substrates with a similar affinity. It appears that the existence of these enzymes is the result of convergent evolution. The amino acid sequence of 6-hydroxy-l-nicotine oxidase (EC 1.5.3.6) as derived from the respective structural gene shows considerable structural similarity with eukaryotic monoamine oxidases (EC 1.4.3.4) but not with monoamine oxidases from prokaryotic bacteria including those of the genus Arthrobacter. These similarities are not confined to the nucleotide-binding sites. A 100-amino acid stretch at the N-terminal regions of 6-hydroxy-l-nicotine oxidase and human monoamine oxidases A possess a 35% homology. Overall, 27.0, 26.9, and 25.8% of the amino acid positions of the monoamine oxidases of Aspergillus niger (N), humans (A), and rainbow trout (Salmo gairdneri) are identical to those of 6-hydroxy-l-nicotine oxidase (Smith–Waterman algorithm). In addition, the G+C content of the latter enzyme is in the range of that of eukaryotic monoamine oxidases and definitely lower than that of the A. nicotinovorans DNA and even that of the pAO1 DNA. The primary structure of 6-hydroxy-d-nicotine oxidase (EC 1.5.3.5) does not reveal its evolutionary history as easily. Significant similarities are found with a mitomycin radical oxidase from Streptomyces lavendulae (23.3%) and a ``hypothetical protein' from Mycobacterium tuberculosis (26.0%). It is proposed that the plasmid-encoded gene of 6-hydroxy-l-nicotine oxidase evolved after horizontal transfer from an eukaryotic source. Received: 6 March 1998 / Accepted: 15 July 1998     

Gas-liguid chromatography of parathion metabolites as a sensitive index of induction of liver microsomal mixed-function oxidases

A method for the gas-liquid chromatographic evaluation of induction of liver microsomal mixed-function oxidases is described. The sensitivity and reproducibility of this method allows statistically significant comparisons of inducers in small numbers of animals, or, by using larger treatment groups, should allow evaluation of minimal levels of inducer. The method is compared to several other indices of mixed-function oxidase induction in rats pretreated with DDT or DDE. The induction of squirrel monkey liver microsomal mixed-function oxidases by the drug diphenylhydantoin has also been demonstrated using this procedure.