Bacterial Utilization of Ether Glycols

A soil bacterium capable of using oligo- and polyethylene glycols and ether alcohols as sole sources of carbon for aerobic growth was isolated. The effects of substituent groups added to the ether bonds on the acceptability of the compounds as substrates were studied. Mechanisms for the incorporation of two-carbon compounds were demonstrated by the observation that acetate, glyoxylate, ethylene glycol, and a number of the tricarboxylic acid cycle intermediates served as growth substrates in minimal media. The rate of oxidation of the short-chained ethylene glycols by adapted resting cells varied directly with increasing numbers of two-carbon units in the chains from one to four. The amount of oxygen consumed per carbon atom of oligo- and polyethylene glycols was 100% of theoretical, but only 67% of theoretical for ethylene glycol. Resting cells oxidized oligo- and polyethylene glycols with 2 to 600 two-carbon units in the chains. Longer chained polyethylene glycols (up to 6,000) were oxidized at a very slow rate by these cells. Dehydrogenation of triethylene glycol by adapted cells was observed, coupling the reaction with methylene blue reduction.     

Metabolism of polyethylene glycol by two anaerobic bacteria, Desulfovibrio desulfuricans and a Bacteroides sp

Two anaerobic bacteria were isolated from polyethylene glycol (PEG)-degrading, methanogenic, enrichment cultures obtained from a municipal sludge digester. One isolate, identified as Desulfovibrio desulfuricans (strain DG2), metabolized oligomers ranging from ethylene glycol (EG) to tetraethylene glycol. The other isolate, identified as a Bacteroides sp. (strain PG1), metabolized diethylene glycol and polymers of PEG up to an average molecular mass of 20,000 g/mol [PEG 20000; HO-(CH2-CH2-O-)nH]. Both strains produced acetaldehyde as an intermediate, with acetate, ethanol, and hydrogen as end products. In coculture with a Methanobacterium sp., the end products were acetate and methane. Polypropylene glycol [HO-(CH2-CH2-CH2-O-)nH] was not metabolized by either bacterium, and methanogenic enrichments could not be obtained on this substrate. Cell extracts of both bacteria dehydrogenated EG, PEGs up to PEG 400 in size, acetaldehyde, and other mono- and dihydroxylated compounds. Extracts of Bacteroides strain PG1 could not dehydrogenate long polymers of PEG (greater than or equal to 1,000 g/mol), but the bacterium grew with PEG 1000 or PEG 20000 as a substrate and therefore possesses a mechanism for PEG depolymerization not present in cell extracts. In contrast, extracts of D. desulfuricans DG2 dehydrogenated long polymers of PEG, but whole cells did not grow with these polymers as substrates. This indicated that the bacterium could not convert PEG to a product suitable for uptake.     

Metabolism of polyethylene glycol by two anaerobic bacteria, Desulfovibrio desulfuricans and a Bacteroides sp.

Two anaerobic bacteria were isolated from polyethylene glycol (PEG)-degrading, methanogenic, enrichment cultures obtained from a municipal sludge digester. One isolate, identified as Desulfovibrio desulfuricans (strain DG2), metabolized oligomers ranging from ethylene glycol (EG) to tetraethylene glycol. The other isolate, identified as a Bacteroides sp. (strain PG1), metabolized diethylene glycol and polymers of PEG up to an average molecular mass of 20,000 g/mol [PEG 20000; HO-(CH2-CH2-O-)nH]. Both strains produced acetaldehyde as an intermediate, with acetate, ethanol, and hydrogen as end products. In coculture with a Methanobacterium sp., the end products were acetate and methane. Polypropylene glycol [HO-(CH2-CH2-CH2-O-)nH] was not metabolized by either bacterium, and methanogenic enrichments could not be obtained on this substrate. Cell extracts of both bacteria dehydrogenated EG, PEGs up to PEG 400 in size, acetaldehyde, and other mono- and dihydroxylated compounds. Extracts of Bacteroides strain PG1 could not dehydrogenate long polymers of PEG (greater than or equal to 1,000 g/mol), but the bacterium grew with PEG 1000 or PEG 20000 as a substrate and therefore possesses a mechanism for PEG depolymerization not present in cell extracts. In contrast, extracts of D. desulfuricans DG2 dehydrogenated long polymers of PEG, but whole cells did not grow with these polymers as substrates. This indicated that the bacterium could not convert PEG to a product suitable for uptake.     

Xenoestrogenic short ethoxy chain nonylphenol is oxidized by a flavoprotein alcohol dehydrogenase from <Emphasis Type="Italic">Ensifer</Emphasis> sp. strain AS08

The ethoxy chains of short ethoxy chain nonylphenol (NPEO_av2.0, containing average 2.0 ethoxy units) were dehydrogenated by cell-free extracts from Ensifer sp. strain AS08 grown on a basal medium supplemented with NPEO_av2.0. The reaction was coupled with the reduction in 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide and phenazine methosulfate. The enzyme (NPEO_av2.0 dehydrogenase; NPEO-DH) was purified to homogeneity with a yield of 20% and a 56-fold increase in specific activity. The molecular mass of the native enzyme was 120 kDa, consisting of two identical monomer units (60 kDa). The gene encoding NPEO-DH was cloned, which consisted of 1,659 bp, corresponding to a protein of 553 amino acid residues. The deduced amino acid sequence agreed with the N-terminal amino acid sequence of the purified NPEO-DH. The presence of a flavin adenine dinucleotide (FAD)-binding motif and glucose–methanol–choline (GMC) oxidoreductase signature motifs strongly suggested that the enzyme belongs to the GMC oxidoreductase family. The protein exhibited homology (40–45% identity) with several polyethylene glycol dehydrogenases (PEG-DHs) of this family, but the identity was lower than those (approximately 58%) among known PEG-DHs. The substrate-binding domain was more hydrophobic compared with those of glucose oxidase and PEG-DHs. The recombinant protein had the same molecular mass as the purified NPEO-DH and dehydrogenated PEG400-2000, NPEO_av2.0 and its components, and NPEOav10, but only slight or no activity was found using diethylene glycol, triethylene glycol, and PEG200. English edition: The paper was edited by a native speaker through American Journal Experts ().     

Microbial Degradation of Polyethylene Glycols

Mono-, di-, tri-, and tetraethylene glycols and polyethylene glycols (PEG) with molecular weight up to 20,000 were degraded by soil microorganisms. A strain of Pseudomonas aeruginosa able to use a PEG of average molecular weight 20,000 was isolated from soil. Washed cells oxidized mono and tetraethylene glycols, but O₂ consumption was not detectable when such cells were incubated for short periods with PEG 20,000. However, the bacteria excreted an enzyme which converted low- and high-molecular-weight PEG to a product utilized by washed P. aeruginosa cells. Gas chromatography of the supernatant of a culture grown on PEG 20,000 revealed the presence of a compound co-chromatographing with diethylene glycol. A metabolite formed from PEG 20,000 by the extracellular enzyme preparation was identified as ethylene glycol by combined gas chromatography-mass spectrometry.     

Degradation of benzenesulfonate to sulfite in bacterial extract.

Sulfite formation from benzenesulfonate was studied in extracts from a bacterium grown on this compound as a main carbon source. The activity of sulfite formation depended on the presence of NADH and oxygen as well as magnesium, suggesting an oxygenation-type reaction. The activity was found in a fraction precipitated by ammonium sulfate at 35-50% saturation; the specific activity was 15 times higher than that of crude extract, probably due to the elimination of inhibitory substances of low molecular weight from the preparation. In a distillate of the reaction mixture, phenol was found. Pyrocatechol as well as benzenesulfonate was oxidized in the crude extract, but phenol was not.     

DEGRADATION OF PYRUVATE BY MICROCOCCUS LACTILYTICUS II. : Studies of Cofactors in the Formate-Exchange Reaction.

McCormick, N. G. (University of Washington, Seattle), E. J. Ordal, and H. R. Whiteley. Degradation of pyruvate by Micrococcus lactilyticus. II. Studies of cofactors in the formate-exchange reaction. J. Bacteriol. 83:899-906. 1962.-Enzyme preparations from Micrococcus lactilyticus(2) are rendered inactive with respect to formate exchange by treatment with charcoal or Dowex-50, by dialysis, or by fractionation with ammonium sulfate. The activity may be completely restored by a "kochsaft" preparation (BES) obtained from M. lactilyticus and partially restored by similar BES preparations from Escherichia coli and Clostridium butyricum. Diphosphothiamine is required for formate exchange but full activity cannot be restored by known cofactors. Brief exposure to increased temperatures, air, extremes of pH, and absorption with charcoal and Dowex-50 decrease the cofactor activity of BES preparations. The addition of BES preparations from E. coli and Streptococcus faecalis causes a shift in the degradation of pyruvate by extracts of M. lactilyticus from the phosphoroclastic cleavage (to acetyl phosphate and formate) to the dismutation of pyruvate (to lactate, acetate, and carbon dioxide).C. cylindrosporum was found to mediate the formate-exchange reaction; the activity of crude extracts was stimulated by M. lactilyticus and C. butyricum BES preparations. M. lactilyticus BES also increased the formate-exchange activity of extracts of E. coli.     

Metabolic pathway of xenoestrogenic short ethoxy chain-nonylphenol to nonylphenol by aerobic bacteria, Ensifer sp. strain AS08 and Pseudomonas sp. strain AS90

Ensifer sp. strain AS08 and Pseudomonas sp. strain AS90 degrading short ethoxy (EO) chain-nonylphenol (NP) [NPEO_av2.0 containing NP mono- ∼ tetraethoxylates (NP1EO ∼ NP4EO); average 2.0 EO units] were isolated by enrichment cultures. Both strains grew on NP but not on octyl- and nonylphenol polyethoxylates (NPEOs) (average 10 EO units). Growth and degradation of NPEO_av2.0 was increased with increased concentrations of yeast extract (0.02–0.5%) in a culture medium. Culture supernatants of both strains grown on NPEO_av2.0 were analyzed by high-performance liquid chromatography, showing degradation of NP4EO–NP1EO. The metabolites from nonylphenol diethoxylate (NP2EO) by resting cells of both strains were identified by gas chromatography–mass spectrometry as nonylphenoxyethoxyacetic acid, NP1EO, nonylphenoxyacetic acid (NP1EC), and NP, while those from NP1EO were identified as NP1EC and NP. Cell-free extracts from strain AS08 grown on NPEO_av2.0 dehydrogenated NPEOs, NPEO_av2.0, NP2EO, NP1EO, and PEG 400, but the extracts were inactive toward di- ∼ tetraethylene glycol. Aldehydes were formed in the reaction mixture of each substrate with cell-free extracts. From these results, the aerobic metabolic pathway for short EO chain-NP is proposed: A terminal alcohol group of the EO chain is oxidized to a carboxylic acid via an aldehyde, and then one EO unit is removed. This process is repeated until NP is produced.English edition: The paper was edited by a native speaker through KN international ()     

Purification and characterization of polyethylene glycol dehydrogenase involved in the bacterial metabolism of polyethylene glycol

Polyethylene glycol (PEG) dehydrogenase in crude extracts of a PEG 20,000-utilizing mixed culture was purified 24 times by precipitation with ammonium sulfate, solubilization with laurylbetaine, and chromatography with diethylamino-ethyl-cellulose, hydroxylapatite, and Sephadex G-200. The purified enzyme was confirmed to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme, which appeared to consist of four identical subunits, was 2.4 X 10(5). The enzyme was stable below 35 degrees C and in the pH range of 7.5 to 9.0. The optimum pH and temperature of the activity were around 8.0 and 60 degrees C, respectively. The enzyme did not require any metal ions for activity and oxidized various kinds of PEGs, among which PEG 6,000 was the most active substrate. The apparent Km values for tetraethylene glycol and PEG 6,000 were about 10.0 and 3.0 mM, respectively.     

Purification and characterization of polyethylene glycol dehydrogenase involved in the bacterial metabolism of polyethylene glycol.

Polyethylene glycol (PEG) dehydrogenase in crude extracts of a PEG 20,000-utilizing mixed culture was purified 24 times by precipitation with ammonium sulfate, solubilization with laurylbetaine, and chromatography with diethylamino-ethyl-cellulose, hydroxylapatite, and Sephadex G-200. The purified enzyme was confirmed to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme, which appeared to consist of four identical subunits, was 2.4 X 10(5). The enzyme was stable below 35 degrees C and in the pH range of 7.5 to 9.0. The optimum pH and temperature of the activity were around 8.0 and 60 degrees C, respectively. The enzyme did not require any metal ions for activity and oxidized various kinds of PEGs, among which PEG 6,000 was the most active substrate. The apparent Km values for tetraethylene glycol and PEG 6,000 were about 10.0 and 3.0 mM, respectively.     

Regioselective synthesis of ethoxylated glycoside esters using beta-glucosidase in supersaturated solutions and lipases in organic solvents

Three ethoxylated glycosides, tetraethylene glycol beta-D-glucoside, tetraethylene glycol beta-D-xyloside, and methoxy triethyleneglycol beta-D-glucoside, were prepared via almond beta-glucoside-catalyzed (trans)glycosylation carried out in supersaturated solutions of glucose or p-nitrophenyl beta-D-xyloside and the respective polyethylene glycols. The products were isolated and further modified by enzymatic esterification with Candida antarctica and Mucor miehei lipases. The latter enzyme showed a much greater selectivity for the primary hydroxyl group on the polyethylene glycol chain of the glucoside substrate, thus enabling us to obtain exclusively the corresponding monoester, omega-O-oleoyl tetraethylene glycol beta-D-glucoside. Novozyme was used for the preparative synthesis of two other monoesters, 6-O-oleoyl (methoxy triethyleneglycol) beta-D-glucoside and omega-O-oleoyl tetraethylene glycol beta-D-xyloside. Two diesters, di-oleoyl tetraethylene glycol beta-D-glucoside and tetraethylene-bis(6-0-oleoyl glucoside) were also synthesized in good yields using this lipase. Copyright 1998 John Wiley & Sons, Inc.     

Ether-cleaving enzyme and diol dehydratase involved in anaerobic polyethylene glycol degradation by a new Acetobacterium sp.

A strictly anaerobic, homoacetogenic bacterium was enriched and isolated from anoxic sewage sludge with polyethylene glycol (PEG) 1000 as sole source of carbon and energy, and was assigned to the genus Acetobacterium on the basis of morphological and physiological properties. The new isolate fermented ethylene glycol and PEG's with molecular masses of 106 to 1000 to acetate and small amounts of ethanol. The PEG-degrading activity was not destroyed by proteinase K treatment of whole cells. In cell-free extracts, a diol dehydratase and a PEG-degrading (ether-cleaving) enzyme activity were detected which both formed acetaldehyde as reaction product. The diol dehydratase enzyme was oxygen-sensitive and was stimulated 10–14 fold by added adenosylcobalamine. This enzyme was found mainly in the cytoplasmic fraction (65%) and to some extent (35%) in the membrane fraction. The ether-cleaving enzyme activity reacted with PEG's of molecular masses of 106 to more than 20000. The enzyme was measurable optimally in buffers of high ionic strength (4.0), was extremely oxygen-sensitive, and was inhibited by various corrinoids (adenosylcobalamine, cyanocobalamine, hydroxocobalamine, methylcobalamine). This enzyme was found exclusively in the cytoplasmic fraction. It is concluded that PEG is degraded by this bacterium inside the cytoplasm by a hydroxyl shift reaction, analogous to a diol dehydratase reaction, to form an unstable hemiacetal intermediate. The name polyethylene glycol acetaldehyde lyase is suggested for the responsible enzyme.Abbreviations EG ethylene glycol - DiEG diethylene glycol - TriEG triethylene glycol - TeEG tetraethylene glycol - PEG polyethylene glycol (molecular mass indicated)     

Purification and characterization of dihydropyrimidine dehydrogenase from pig liver

Dihydropyrimidine dehydrogenase was isolated from cytosolic pig liver extracts and purified 3100-fold to apparent homogeneity. Purification made use of ammonium sulfate fractionation, precipitation with acetic acid and chromatography on DEAE-cellulose and 2',5'-ADP-Sepharose with 28% recovery of total activity. The native enzyme has a molecular mass of 206 kDa and is apparently composed of two similar, if not identical, subunits. Proteolytic cleavage reveals two fragments with apparent molecular masses of 92 kDa and 12 kDa. The C-terminal 12-kDa fragment seems to be extremely hydrophobic. The enzyme contains tightly associated compounds including four flavin nucleotide molecules and 32 iron atoms/206-kDa molecule. The iron atoms are probably present in iron-sulfur centers. The flavins released from the enzyme were identified as FAD and FMN in equal amounts. An isoelectric point of 4.65 was determined for the dehydrogenase. Apparent kinetic parameters were obtained for the substrates thymine, uracil, 5-aminouracil, 5-fluorouracil and NADPH.     

Initial Stages in the Biodegradation of the Surfactant Sodium Dodecyltriethoxy Sulfate by Pseudomonas sp. Strain DES1

The biodegradation of the surfactant sodium dodecyltriethoxy sulfate by Pseudomonas sp., strain DES1 (isolated from activated sludge plant effluent) has been studied. Growth of the organism when the ³⁵S-labeled surfactant was present as the sole source of carbon and energy led to the appearance in the culture fluid of five ³⁵S-labeled organic metabolites. These have been identified as mono-, di-, and triethylene glycol monosulfates (major metabolites) and acetic acid 2-(ethoxy sulfate) and acetic acid 2-(diethoxy sulfate), authentic samples of which have been prepared and characterized. Evidence is presented that the major metabolites were produced by rupture of one or another of the three ether linkages present in the surfactant molecule, probably via the agency of a single etherase enzyme. Acetic acid 2-(ethoxy sulfate) and acetic acid 2-(diethoxy sulfate) were formed by the oxidation of the free alcohol groups of di- and triethylene glycol monosulfates, respectively, and increased in amount during the stationary phase of growth. Inorganic ³⁵S-sulfate also appeared in significant quantities in culture fluids and arose from the parent surfactant (presumably via the action of an alkylsulfatase) and not from any of the five metabolites. The appearance of sulfated organic metabolites during the exponential phase of growth and their quantitative relationship remained remarkably constant, even when additional carbon and energy sources (succinate or yeast extract) were also present in the growth media.     

Muscle-derived factors that support survival and promote fiber outgrowth from embryonic chick spinal motor neurons in culture

The purposes of the experiments reported is to provide an unambiguous demonstration that embryonic skeletal muscle contains factors that act directly on embryonic spinal motor neurons both to support their survival and to stimulate the outgrowth of neurites. Cells of lumbar and brachial ventral spinal cords from 6-day-old chick embryos were separated by centrifugation in a two-step metrizamide gradient, and a motor neuron enriched fraction was obtained. Motor neurons were identified by retrogradely labeling with rhodamine isothiocyanate, and were enriched fourfold in the motor neuron fraction relative to unfractionated cells. In culture, the isolated motor neurons died within 3-4 days unless they were supplemented with embryonic chick skeletal muscle extract. Two functionally distinct entities separable by ammonium sulfate precipitation were responsible for the effects of muscle extracts on motor neurons. The 0-25% ammonium sulfate precipitate contained molecules that alone had no effect on neuronal survival but when bound to polyornithine-coated culture substrata, stimulated neurite outgrowth and potentiated the survival activity present in muscle. Most of this activity was due to a laminin-like molecule being immunoprecipitated with antisera against laminin, and immunoblotting demonstrated the presence of both the A and B chains of laminin. A long-term survival activity resided in the 25-70% ammonium sulfate fraction, and its apparent total and specific activities were strongly dependent on the culture substrate. In contrast to the motor neurons, the cells from the other metrizamide fraction (including neuronal cells) could be kept in culture for a prolonged time without addition of exogenous factor(s).     

Existence of ether bond-cleaving enzyme in a polyethylene glycol-utilizing symbiotic mixed culture

Abstract Diglycolic acid dehydrogenase activity linked with 2,6-dichlorophenolindophenol and phenazine methosulfate was found in the particulate fraction of the cell-free extract of a mixed culture of Flavobacterium and Pseudomonas species grown on polyethylene glycol 6000. The amount of glyoxylic acid formed increased with the increase in reaction time and enzyme concentration. Horse heart cytochrome c , 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl 2H-tetrazolium bromide, and nitro blue tetrazolium, served as hydrogen acceptors in the presence of phenazine methosulfate. Enzyme activity was competitively inhibited by 1,4-benzoquinone. The enzyme was also active on tetraethylene glycol dicarboxylic acid, a metabolite of tetraethylene glycol, and on methoxy- or ethoxyacetic acid.     

The in vivo transformation of phospholipid vesicles to a particle resembling HDL in the rat.

The tumor-specific transplantation antigen (TSTA) in crude three molar potassium chloride (3M KCl) extracts of a chemically-induced, murine fibrosarcoma was purified by ammonium sulfate salt fraction at 20% saturation (S20S) and by polyacrylamide gel electrophoresis (PAGE). Mice, which had been pretreated with the S20S precipitate, displayed retarded outgrowth of a 100-fold supralethal dose of the corresponding, but not of a non-cross-reactive syngeneic tumor. Analysis of PAGE gels by Coomassie Blue staining revealed at least 30 bands in the crude 3M KCl extract, and only two components (Rf 0.34 and 0.43) in the ammonium sulfate fraction. That these two components bore TSTA activity was demonstrated by the observation that the immunoprotective activity of crude 3M KCl extracts was localized to the Rf 0.25–0.50 region. The two components present in the S20S fraction had isoelectric points of 5.05 and 6.9, and estimated molecular weights of 40,000 and 75,000, thus demonstrating the soluble nature of the active principle. These findings offer the prospect of a chemical dissection of the polymorphic TSTA surface markers on MCA-induced murine tumors.     

Marinobacterium sp. strain DMS-S1 uses dimethyl sulphide as a sulphur source after light-dependent transformation by excreted flavins

Marinobacterium sp. strain DMS-S1 is a unique marine bacterium that can use dimethyl sulphide (DMS) as a sulphur source only in the presence of light. High-performance liquid chromatography (HPLC) analyses of the culture supernatant revealed that excreted factors, which could transform DMS to dimethyl sulphoxide (DMSO) under light, are FAD and riboflavin. In addition, FAD appeared to catalyse the photolysis of DMS to not only DMSO but also methanesulphonate (MSA), formate, formaldehyde and sulphate. As strain DMS-S1 can use sulphate and MSA as a sole sulphur source independently of light, the excretion of flavins appeared to support the growth on DMS under light. Furthermore, three out of 12 marine bacteria from IAM culture collection were found to be able to grow on DMS with the aid of photolysis by the flavins excreted. This is the first report that bacteria can use light to assimilate oceanic organic sulphur compounds outside the cells by excreting flavins as photosensitizers.     

An inhibitor of protein synthesis in cytoplasmic extracts of density inhibited cells

Cytoplasmic extracts of green monkey kidney-Vero M3 cells that have been grown to high cell density and have entered the stationary phase of growth lose the capacity to synthesize proteins after they have been frozen and thawed. The same loss of protein synthetic capacity is not observed when cytoplasmic extracts of low cell density Vero M3 or HeLa S-3 cells are frozen and thawed before assay, nor when high cell density Vero M3 cell extract is assayed without having been frozen. The loss of the protein synthesis capacity of the frozen and thawed extracts of stationary phase Vero M3 cells is accounted for by the appearance of an inhibitor. The inhibitor is precipitated in the 30 to 70% ammonium sulfate fraction of the 100,000 g supernatant. It is inactivated by heat and is non-dialyzable. It blocks the transfer of amino acids from tRNA to growing peptide chains. The amount of this inhibitor in the extract (measured by relative inhibition of in vitro protein synthesis) increases as a function of the density of the cells from which the extract was made, and the increase can be correlated with the progressive turnoff of protein synthesis in whole cells.     

Reduced nicotinamide adenine dinucleotide oxidase activity and H2O2 formation of Mycoplasma pneumoniae

Cell-free extracts of Mycoplasma pneumoniae showed two distinct reduced nicotinamide adenine dinucleotide (NADH(2)) oxidase activities in the supernatant fraction. By ammonium sulfate fractionation and polyacrylamide gel electrophoresis, one activity not requiring flavine co-factors was precipitated by 50 to 70% ammonium sulfate concentration and identified with a slower-moving band on acrylamide gel electrophoresis; a second NADH(2) oxidase activity was flavine mononucleotide (FMN) dependent and associated with a more rapidly moving band; it could only be partially precipitated by ammonium sulfate concentrations ranging from 50 to 100%. Studies with alternate electron acceptors indicated the presence of a menadione, a 2,6-dichlorophenol indophenol and a very weak ferricyanide oxido-reductase activity, but no cytochrome c oxido-reductase, in the cell-free preparations. The NADH(2) oxidase activities of all fractions were relatively cyanide insensitive and were only minimally inhibited by flavoprotein and other respiratory chain inhibitors. H(2)O(2) formation was negligible unless FMN, but not flavine adenine dinucleotide (FAD), was added to the crude NADH(2) oxidase system; upon fractionation and electrophoresis, the H(2)O(2) formation was associated with the FMN-dependent, more rapidly moving NADH(2) oxidase band. This FMN-dependent NADH(2) oxidase-H(2)O(2) generating system may be a mechanism for the H(2)O(2) formation observed during glucose oxidation in the intact organism.