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Query: UMLS:C0027960 (mole)
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Two aldose (xylose) reductases (ARI and ARII) from Fusarium oxysporum were purified and characterized. The native ARI was a monomer with M(r) 41000, pI 5.2 and showed a 52-fold preference for NADPH over NADH, while ARII was homodimeric with a subunit of M(r) 37000, pI 3.6 and a 60-fold preference for NADPH over NADH. In this study, the influence of aeration and the response to the addition of electron acceptors on xylose fermentation by F. oxysporum were also studied. The batch cultivation of F. oxysporum on xylose was performed under aerobic, anaerobic and oxygen-limited conditions in stirred tank reactors. Oxygen limitation had considerable influence on xylose metabolism. Under anaerobic conditions (0 vvm), xylitol was the main product with a maximum yield of 0.34 mole of xylitol/mole of xylose while the maximum ethanol yield (1.02 moles of ethanol/mole of xylose) was obtained under aerobic conditions (0.3 vvm). When the artificial electron acceptor acetoin was added to an anaerobic batch fermentation of xylose by F. oxysporum, the ethanol yield increased while xylitol excretion was also decreased.
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PMID:NADPH-dependent D-aldose reductases and xylose fermentation in Fusarium oxysporum. 1623 33

Pseudomonas cepacia AC1100 degrades 2,4,5-trichlorophenoxyacetate (2,4,5-T), an herbicide and chlorinated aromatic compound. Although some progress has been made in understanding 2,4,5-T degradation by AC1100 by molecular analysis, little is known about the biochemistry involved. Enzymatic activity converting 2,4,5-T to 2,4,5-trichlorophenol in the presence of NADH and O(inf2) was detected in cell extracts of AC1100. Phenyl agarose chromatography of the ammonium sulfate-fractionated cell extracts yielded no active single fractions, but the mixing of two fractions, named component A and component B, resulted in the recovery of enzyme activity. Component B was further purified to homogeneity by hydroxyapatite and DEAE chromatographies. Component B had a native molecular weight of 140,000, and it was composed of two 49-kDa (alpha)-subunits and two 24-kDa (beta)-subunits. Component B was red, and its spectrum in the visible region had maxima at 430 and 560 nm (shoulder), whereas upon reduction it had maxima at 420 (shoulder) and 530 nm. Each mole of (alpha)(beta) heterodimer contained 2.9 mol of iron and 2.1 mol of labile sulfide. These properties suggest strong similarities between component B and the terminal oxygenase components of the aromatic ring-hydroxylating dioxygenases. Component A was highly purified but not to homogeneity. The reconstituted 2,4,5-T oxygenase, consisting of components A and B, converted 2,4,5-T quantitatively into 2,4,5-trichlorophenol and glyoxylate with the coconsumption of NADH and O(inf2).
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PMID:Purification and Properties of Component B of 2,4,5-Trichlorophenoxyacetate Oxygenase from Pseudomonas cepacia AC1100. 1653 34

Ascorbic acid oxidase activity in Myrothecium verrucaria extracts resulted in O(2) uptake exceeding 0.5 mole per mole of ascorbic acid and in CO(2) evolution. Measurement of oxidized ascorbic acid at completion of the reaction demonstrated that an average of 10% of the oxidized product disappeared. A comparison of the gas exchange data with the amount of ascorbic acid not accounted for indicated that the reaction could not be explained by independent oxidase and oxygenase systems. Chromatographic examination of the reaction mixtures identified l-threonic acid. Experiments with ascorbic acid-1-(14)C showed that C-1 was partially decarboxylated during the oxidation. Test of the fungal extracts for enzymes that might explain the deviation from expected stoichiometry showed that phenolase, glutathione reductase, cytochrome oxidase, peroxidase and oxalic decarboxylase were not involved. Addition of azide in concentrations sufficient to block catalase increased excess O(2) consumption about 65%. No enzymes were found that could directly attack oxidized ascorbic acid. H(2)O(2) accumulated during oxidation in azide-blocked systems.The O(2) excess could be explained by assuming the enzyme had peroxidative capacity on a reductant other than ascorbic acid. An intermediate of ascorbic acid oxidation appeared to function as the substrate yielding CO(2) and l-threonic acid on degradation. The increase in excess O(2) utilized in azide-blocked systems and the H(2)O(2) accumulation also were explained by the proposed scheme.Another interpretation would involve production of free radicals during ascorbic acid oxidation. Evidence for this was the ability of extracts to oxidize DPNH in the presence of ascorbic acid. Oxygen radicals formed in such reactions were considered possible agents of degradation of ascorbic acid.
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PMID:Reaction Properties of the Ascorbic Acid Oxidase from Myrothecium verrucaria. 1665 40

NADH-nitrate reductase has been highly purified from leaves of 8-day-old wheat (Triticum aestivum L. cv. Olympic) seedlings by affinity chromatography, using blue dextran-Sepharose 4B. Purification was assessed by polyacrylamide gel electrophoresis. The enzyme was isolated with a specific activity of 23 micromoles nitrite produced per minute per milligram protein at 25 C. At pH 7.5, the optimum pH for stability of NADH-nitrate reductase, this enzyme, and a component enzyme reduced flavin adenine mononucleotide (FMNH(2))-nitrate reductase has a similar stability at both 10 and 25 C. Two other component enzymes-methylviologen-nitrate reductase and NADH-ferricyanide reductase-also have a similar but higher stability. At this pH the Arrhenius plot for decay of NADH-nitrate reductase and methylviologen-nitrate reductase indicates a transition temperature at approximately 30 C above which the energy of activation for denaturation increases. FMNH(2)-nitrate reductase and NADH-ferricyanide reductase do now show this transition. The energy of activation for denaturation (approximately 9 kcal per mole) of each enzyme is similar between 15 and 30 C. The optimum pH for stability of the component enzymes was: NADH-ferricyanide reductase, 6.6; FMNH(2)-nitrate reductase and methylviologen-nitrate reductase, 8.9. All of our studies indicate that the NADH-ferricyanide reductase was the most stable component of the purified nitrate reductase (at pH 6.6, t((1/2)) [25 C] = 704 minutes). Data are presented which suggest that methylviologen and FMNH(2) do not donate electrons to the same site of the nitrate reductase protein.
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PMID:In vitro stability of nitrate reductase from wheat leaves: I. Stability of highly purified enzyme and its component activities. 1666 Jul 26

Spinach (Spinacia oleracea L.) leaf discs accumulated free proline when exposed to polyethylene glycol solutions of water potential less than -10 bars. At -20 bars, the accumulation was 11 micromoles per gram original fresh weight in a 24-hour period.When the leaf organelles were separated on a sucrose gradient, a proline oxidase was detected in the mitochondrial fraction. Isolated mitochondria were used for the study of the properties of the enzyme which was assayed by both oxygen uptake measurement and reduction of 2,6-dichlorophenol-indophenol in the presence of phenazine methosulfate. There was a stoichiometry of one-half mole of oxygen uptake per mole of Delta(1)-pyrroline-5-carboxylate production in the enzymic reaction. The enzyme had an optimal activity at pH 8.0 to 8.5 and an apparent K(m) value of 0.028 molar for proline. MgCl(2) and flavin adenine dinucleotide were required for maximal activity. Addition of sucrose, mannitol, or polyethylene glycol to reduce the water potential of the reaction mixture to as low as -20 bars resulted in little inhibition. The enzyme preparation was unable to reduce NAD to NADH, and NAD did not inhibit the enzyme activity. The enzyme preparation reduced cytochrome c in the presence of KCN. Triton X-100 at low concentration strongly inhibited the enzyme activity. The enzyme was apparently linked to the mitochondrial electron transport system. The in vitro activity of the enzyme under optimal assay conditions was high enough to prevent proline accumulation under water stress condition; presumably this activity was restrained in vivo.
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PMID:Proline Oxidase and Water Stress-induced Proline Accumulation in Spinach Leaves. 1666 Jul 61

A plasmalemma-bound NADH oxidation system (Lin 1982 Proc Natl Acad Sci USA 79: 3773-3776) in corn root protoplasts was isolated by a mild treatment of intact protoplasts with trypsin. The majority of NADH stimulated O(2) consumption activity of the protoplasts could be recovered in the supernatant isolated from the intact protoplasts which have been treated with trypsin. The activation energy of NADH oxidation in the supernatant is similar to that of the intact protoplasts (8.7 versus 9.4 kilocalories per mole per degree). Unlike that of the intact protoplasts, an Arrhenius plot of the temperature response (from 5 to 25 degrees C) of the activity in the supernatant shows no transition suggestive of a dissociation of the enzyme from the membrane. Trypsin treatment did not affect K(+) uptake into cell volume of the protoplast. However, the NADH-stimulated K(+) uptake and the increase of cell volume were greatly reduced. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of trichloroacetic acid-precipitated protein from the supernatant showed one extra peptide band with approximately 42 kilodalton molecular weight.
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PMID:Isolation of NADH Oxidation System from the Plasmalemma of Corn Root Protoplasts. 1666 74

The isotherm for isocitrate oxidation by potato (Solanum tuberosum L. var. Russet Burbank) mitochondria in the presence of exogenous NAD is characterized by a hyperbolic phase at isocitrate concentrations below 3 millimolar, and a sigmoid, or positively cooperative phase from approximately 3 to 30 millimolar. The two forms of isocitrate dehydrogenase were separated and characterized following the sonication of mitochondria in 15% glycerol in the absence of buffer, followed by fractionation in a density step gradient to yield inner membrane and matrix components. The membrane-associated isocitrate dehydrogenase was found to have a Hill, or cooperativity, number of 1, while the Hill number of the matrix enzyme was 2.5. Upon digitonin extraction the cooperativity number of the membrane enzyme rose to 3.5. The isocitrate K(m) for the membrane enzyme was calculated to be approximately 5.9 x 10(-4) molar, while the S(0.5) for the matrix was 6.9 x 10(-4) molar. The NAD K(m) for both enzymes was 150 micromolar. Whereas the membrane enzyme proved indifferent to adenine nucleotides, the matrix enzyme was arguably inhibited by AMP and ADP, and inhibited some 25% by 5 millimolar ATP. Both enzymes were negatively responsive to the mole fraction of NADH, the membrane enzyme being 50% inhibited at a mole fraction of 0.26, and the matrix enzyme by a mole fraction of 0.32. The suggestion is offered that the enzymes in question constitute two forms of a single enzyme, one peripherally associated with the inner membrane, and one soluble in the matrix. It is proposed that a degree of regulation may be achieved by the apportionment of the enzyme between the bound and free forms.
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PMID:Isolation and Characterization of Inner Membrane-Associated and Matrix NAD-Specific Isocitrate Dehydrogenase in Potato Mitochondria. 1666 46

Enthalpy changes in the formation of a proton electrochemical potential (Delta mu H+) and its components, DeltapH (proton gradient) and Deltapsi (electrical potential), across two types of E. coli membrane vesicles were investigated. Flow dialysis experiments showed that in 0.1 M KPi, pH 6.6, E. coli GR19N membrane vesicles coupled with d-lactate exhibited 57 mV for DeltapH, 70 mV for Deltapsi, and 127 mV for Delta mu H+. Microcalorimetric measurements revealed that the corresponding enthalpy changes (DeltaH(pH), DeltaH(psi) and DeltaHm) were 3.5, 3.3 and 6.9 kcal/mole, respectively. Moreover, in E. coli ML 308-225 membrane vesicles across which 120mV of Delta mu H+ was generated, values of DeltaH(pH) and DeltaH(psi) were determined as 7.0 and 6.6 kcal/mole, as compared with the previously reported 14.1 kcal/mole for DeltaH(m). Comparisons of these enthalpy data revealed that component enthalpies (DeltaH(pH) and DeltaH(psi)) essentially added up to the total enthalpy (DeltaHm), providing a self-consistent test for the obtained data. In both membranes, the ratio ofDeltaH(psi) to Deltapsi was comparable to that of DeltaH(pH) to DeltapH in the formation of Delta mu H+. These observations indicated that the process of the movement of H+ across the membranes was the major contributor to the observed energetic changes. Moreover, the enthalpy change in the formation of Delta mu H+ was compared with the membranes derived from GR19N and ML 308-225 and coupled with NADH and d-lactate. The results were discussed in terms of trans-membrane phenomena.
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PMID:Enthalpy changes in the formation of the proton electrochemical potential and its components. 1702 Aug 51

Pseudomonas sp. strain C4 metabolizes carbaryl (1-naphthyl-N-methylcarbamate) as the sole source of carbon and energy via 1-naphthol, 1,2-dihydroxynaphthalene, and gentisate. 1-Naphthol-2-hydroxylase (1-NH) was purified 9.1-fold to homogeneity from Pseudomonas sp. strain C4. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme is a homodimer with a native molecular mass of 130 kDa and a subunit molecular mass of 66 kDa. The enzyme was yellow, with absorption maxima at 274, 375, and 445 nm, indicating a flavoprotein. High-performance liquid chromatography analysis of the flavin moiety extracted from 1-NH suggested the presence of flavin adenine dinucleotide (FAD). Based on the spectral properties and the molar extinction coefficient, it was determined that the enzyme contained 1.07 mol of FAD per mol of enzyme. Although the enzyme accepts electrons from NADH, it showed maximum activity with NADPH and had a pH optimum of 8.0. The kinetic constants K(m) and V(max) for 1-naphthol and NADPH were determined to be 9.6 and 34.2 microM and 9.5 and 5.1 micromol min(-1) mg(-1), respectively. At a higher concentration of 1-naphthol, the enzyme showed less activity, indicating substrate inhibition. The K(i) for 1-naphthol was determined to be 79.8 microM. The enzyme showed maximum activity with 1-naphthol compared to 4-chloro-1-naphthol (62%) and 5-amino-1-naphthol (54%). However, it failed to act on 2-naphthol, substituted naphthalenes, and phenol derivatives. The enzyme utilized one mole of oxygen per mole of NADPH. Thin-layer chromatographic analysis showed the conversion of 1-naphthol to 1,2-dihydroxynaphthalene under aerobic conditions, but under anaerobic conditions, the enzyme failed to hydroxylate 1-naphthol. These results suggest that 1-NH belongs to the FAD-containing external flavin mono-oxygenase group of the oxidoreductase class of proteins.
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PMID:Purification and characterization of 1-naphthol-2-hydroxylase from carbaryl-degrading Pseudomonas strain c4. 1723 79

Carbohydrate and fatty acids are major energetic substrates, although amino acid oxidation also permits ATP synthesis. Among several major metabolic differences between lipids and carbohydrate (activation, transport, effect of insulin, etc.), two are of particular importance when considering energy metabolism of critically ill patients: the yield of ATP synthesis and the response to uncoupling. (I) Oxidative phosphorylation yield is higher when NADH is the electron donor (three coupling sites: complex 1, 3 and 4) as compared to FADH2 (two coupling sites: complex 3 and 4). Since the ratio NADH/FADH2 is higher for glycolysis as compared to beta-oxidation, the stoichiometry of ATP synthesis to oxygen consumption is also higher. Lipid oxidation provides more ATP than carbohydrate, but it requires more oxygen per mole of ATP synthesized. (II) The ratio of NADH oxidation versus FADH, oxidation depends on the proton motive force, and lowering proton motive force by uncoupling favours FADH2 oxidation, i.e. lipids versus carbohydrate. In conclusion, lipid oxidation provides a high rate of ATP synthesis even during a mild uncoupling state, but at a high rate of oxygen consumption. If oxygen availability is limited, the major metabolic adaptation to increase the efficiency is represented by a switch from lipid oxidation to glucose oxidation.
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PMID:Choosing the right substrate. 1738 Jul 91


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