Reduced nicotinamide adenine dinucleotide oxidase activity in membranes and cytoplasm of Acholeplasma laidlawii and Mycoplasma mycoides subsp. capri.

The properties of the membrane-bound reduced nicotinamide adenine dinucleotide (NADH) oxidase of Acholeplasma laidlawii were compared with those of the corresponding cytoplasmic activity of Mycoplasma mycoides subsp. capri. The striking differences in pH optima, susceptibility to inhibitors and detergents, and heat inactivation between the NADH oxidase activity, with oxygen as an electron acceptor, and the NADH oxidoreductase activity, with dichlorophenol indophenol (DCPIP) as an alternate electron acceptor, support the presence of more than one catalytic protein in both the membrane-bound and soluble enzyme systems. The detection of more than one band positive for the NADH-nitroblue tetrazolium oxidoreductase reaction on electrophoresis of either the membranes of A. laidlawii or the cytoplasm of M mycoides subsp. capri also points in the same direction. The membrane-bound enzyme system differed, however, form the soluble one because it had a lower ratio of oxidase activity to oxidoreductase activity, and because it was less susceptible to heat inactivation and more readily incorporated incorporated into reaggregated membranes. In addition, the specific activity of the membrane-bound enzyme system increased as the culture aged, whereas that of the soluble system decreased as the culture aged. It is suggested that the different location in the cell could be responsible for some of the differences between the membrane-bound NADH oxidase activity of A. laidlawii and that found in the cytoplasm of M. mycoides subsp. capri.     

Separation of the primary dehydrogenase from the cytochromes of the nicotinamide adenine dinucleotide (reduced form) oxidase of Bacillus megaterium

A selective extraction procedure was developed for sequentially extracting a fraction containing the primary dehydrogenase and a fraction containing the cytochromes of the nicotinamide adenine dinucleotide (reduced form) (NADH) oxidase of Bacillus megaterium KM membranes. The primary dehydrogenase (NADH-2,6-dichlorophenolindophenol oxidoreductase) activity was extracted from sonically treated membranes with 0.4% sodium deoxycholate for 30 min at 4 C. The insoluble residue was extracted with 0.4% sodium deoxycholate in 1 m KCl for 30 min at 25 C. A combination of the two extracts and dilution in Mg(2+) gave good recovery of the original membrane NADH oxidase activity. The primary dehydrogenase fraction contained 41% of the membrane protein, no cytochromes, flavine adenine dinucleotide as the sole acid-extractable flavine, and most of the membrane ribonucleic acid (RNA). The cytochrome-containing fraction had 16% of the membrane protein, 61% of the membrane cytochrome with the same relative amounts of cytochromes a and b as the original membrane, no acid-extractable flavine, little RNA, and no oxidoreductase activity. The oxidoreductase fraction remained soluble after removal of deoxycholate whereas the cytochrome fraction became insoluble after removal of deoxycholate-KCl, but the precipitated fraction could be redissolved in 0.4% sodium deoxycholate. Treatment of both fractions with ribonuclease to destroy all of the RNA present did not affect the ability of the fractions to recombine into a functional oxidase unit. Treatment of either fraction with phospholipase A prevented restoration of a functional oxidase when the oxidoreductase and cytochrome fractions were treated in solution, but no affect on restoration of oxidase was observed when the phospholipase A treatment was carried out with the soluble oxidoreductase fraction and the insoluble cytochrome fraction.     

Adenosine triphosphatase activity of mycoplasma membranes

Rottem, Shlomo (Hebrew University, Jerusalem, Israel), and Shmuel Razin. Adenosine triphosphatase activity of mycoplasma membranes. J. Bacteriol. 92:714-722. 1966.-Adenosine triphosphatase activity of Mycoplasma laidlawii, M. gallisepticum, and Mycoplasma sp. strain 14 was confined to the cell membrane. The enzymatic activity was dependent on magnesium, but was not activated by sodium and potassium. Ouabain did not inhibit the adenosine triphosphatase activity of the mycoplasmas, and did not interfere with the active accumulation of potassium by M. laidlawii cells. Sulfhydryl-blocking reagents and fluoride inhibited the enzymatic activity, whereas 2,4-dinitrophenol was without any effect. Membranes of M. laidlawii hydrolyzed other nucleotide triphosphates and adenosine diphosphate (ADP), but at a lower rate than adenosine triphosphate (ATP). Nucleoside-2'-(3')-phosphates, ribose-5-phosphate, glucose-6-phosphate, and pyrophosphate were not hydrolyzed by the membrane preparations. It seems that the enzyme(s) involved in ATP hydrolysis by M. laidlawii membranes is strongly bound to the membrane subunits, which would account for the failure to purify the enzyme by protein fractionation techniques. The adenosine triphosphatase activity of mycoplasma membranes resembles in its properties that of similar enzymes studied in bacteria. The mycoplasma enzyme(s) seems to differ from the adenosine triphosphatase associated with ion transport in mammalian cell membranes and from mitochondrial adenosine triphosphatase.     

Divalent Cation Activation of Deoxycholate-Solubilized and -Inactivated Membrane Reduced Nicotinamide Adenine Dinucleotide Oxidase of Bacillus megaterium KM

After treating Bacillus megaterium KM membranes with 0.2% sodium deoxycholate, most of the membrane reduced nicotinamide adenine dinucleotide (NADH) oxidase was inactivated, and all of the membrane NADH-2,6 dichlorophenol indophenol oxidoreductase was solubilized. Dilution of the deoxycholate-treated membranes in the presence of divalent cations restored almost all of the original membrane NADH oxidase. The effectiveness of the divalent cation activation decreased in the order Ba(2+) > Ca(2+) > Mg(2+) > Mn(2+). After centrifugation, the deoxycholate-treated membranes at 100,000 x g for 1 hr, all of the NADH oxidase that was activated by a divalent cation was soluble. Cation-activated oxidase, however, was insoluble. The results show that 0.2% deoxycholate at least partially solubilizes the total electron chain from NADH to O(2) in an inactive from which can be reactivated by divalent cations with the formation of active, insoluble NADH oxidase.     

Chemical and Physical Characteristics of the Deoxycholate-Soluble and Magnesium-Reaggregated Membrane Nicotinamade Adenine Dinucleotide (Reduced Form) Oxidase of Bacillus megaterium

The inactive components of the nicotinamide adenine dinucleotide (reduced form) (NADH) oxidase present in the 0.4% deoxycholate-soluble fraction obtained from Bacillus megaterium KM membranes were reaggregated into active NADH oxidase by dilution in the presence of Mg(2+). The reaggregated oxidase was different from the original membrane with respect to sedimentation behavior in a sucrose gradient and morphological appearance. The deoxycholate-insoluble portion of the membrane had membrane-like structure whereas the reaggregated oxidase appeared to be a filamentous aggregate of small particles. The reaggregated oxidase and the deoxycholate-insoluble membrane residue were similar to the original membrane with respect to total protein and total lipid content. The inactive components of the NADH oxidase system exist in deoxycholate as two molecular species which were separable by sucrose density gradient centrifugation or gel filtration in deoxycholate-containing solutions. Both components and dilution in the presence of Mg(2+) were necessary for restoration of oxidase activity. The smaller-molecular-weight component contained all of the NADH-2,6-dichlorophenolindophenol oxidoreductase activity of the original membrane.     

Histochemical demonstration of enzymes related to NADPH-dependent hydroxylating systems in rat liver after phenobarbital treatment

Summary Male rats were given 100mg phenobarbital for three days intraperitoneally. Biochemically an increase was found in activity of nitro-anisole demethylation and in the content of cytochrome P-450. Enzymhistochemically an increase in activity was noted for NADPH tetr. red., G6PD, ICD, and Naftol AS-D-esterase; a decrease was seen in G6Pase and glycogen, but no difference was found in NADH tetr. red. From these results it has been suggested that NADPH tetr. red. is directly involved in the hydroxylation chain, while G6PD and ICD are more indirectly involved.List of Abbreviations NADH nicotinamide adenine dinucleotide - NADPH nicotinamide adenine dinucleotide phosphate - NADPH tetr. red. NADPH tetrazolium reductase - G6PD glucose-6-phosphate dehydrogenase - ICD iso-citric acid dehydrogenase - G6Pase glucose-6-phosphatase - PAS periodic acid-Schiff method     

Fractionation of membrane vesicles from coliphage M13-infected Escherichia coli.

Membrane vesicles were prepared by osmotic lysis of spheroplasts from M13-infected Escherichia coli. Reduced nicotinamide adenine dinucleotide (NADH) oxidase (reduced NAD: oxidoreductase, EC 1.6.99.3) and Mg2+-Ca2+-activated adenosine triphosphatase (ATP phosphohydrolase, EC 3.6.1.3), which are normally localized to the inner surface of the cytoplasmic membrane, were 50% acceesible to their polar substrates in these vesicles. The major coat protein of coliphage M13 is also bound to the cytoplasmic membrane (prior to phage assembly) but with its antigenic sites exposed to the exterior of the cell. Antibody to M13 coat protein was used to fractionate membrane vesicles. Neither agglutinated nor unagglutinated vesicles had altered NADH oxidase and adenosine triphosphatase specific activities. This is inconsistent with such vesicles being a mixture of correctly oriented and completely inverted membrane sacs and suggests that NADH oxidase, adenosine triphosphatase, M13 coat protein, or all three proteins rearrange during vesicle preparation.     

Histochemical demonstration of enzymes in rat liver during post-natal development

Summary Male and female rat liver were studied during post-natal development. A correlation was found between biochemically determined hydroxylations and enzymhisto-chemically determined NADPH-nitro-BT reductase and Naphthol-AS-D esterase. No correlation was found between glucose-6-phosphate dehydrogenase or iso-citric acid dehydrogenase activity and hydroxylations. The difference in hydroxylating capacity between male and female rats may be caused by the fact that the number of cells with hydroxylating activity in the liver lobule, as judged by the NADPH-nitro-BT reductase and Naphthol-AS-D esterase activity, is higher in male than in female rats.List of Abbreviations NADH reduced nicotinamide adenine dinucleotide - NADPH reduced nicotinamide adenine dinucleotide phosphate - G6PD glucose-6-phosphate dehydrogenase - ICD iso-citric acid dehydrogenase - G6Pase glucose-6-phosphatase - NADPH -nitro-BT red - NADPH Nitro-blue tetrazolium reductase - SDH succinic acid dehydrogenase - TCA trichloracetic acid     

Oxidases and reductases are involved in metronidazole sensitivity in Helicobacter pylori

Helicobacter pylori is a contributing factor to the development of gastric and duodenal ulcers and some gastric cancers. Some therapeutic regimes comprise of a number of components, one of which is the antimicrobial metronidazole. A problem with these therapies is the increasing prevalence of metronidazole-resistant (MtrR) H. pylori strains. Several resistance mechanisms have been proposed, and this study addresses the 'scavenging of oxygen' hypothesis. Spectrophotometric assays of cytosolic fractions indicated that metronidazole-sensitive (MtrS) H. pylori isolates had 2.6-fold greater nicotinamide adenine dinucleotide (NADH) oxidase activity, 34-fold greater NADH nitroreductase activity, and eightfold greater nicotinamide adenine dinucleotide phosphate (NADPH) nitroreductase activity than cytosolic fractions from matched MtrR strains. Electrophoresis of cytosolic fractions in non-denaturing gels showed up to 10 protein bands when stained with Coomassie blue. Activity staining of non-denaturing, non-reducing polyacrylamide gels detected NAD(P)H oxidase, disulphide reductase, tetrazolium reductase and nitroreductase activities in the protein bands. Oxidase and reductase activities observed in a band from MtrS strains were absent in the corresponding band from MtrR strains. This band comprised at least 13 proteins, and the major constituent was identified as an alkyl hydroperoxide reductase AhpC subunit. The absence of oxidase and reductase activities in the band from MtrR strains indicated a correlation between the activity of the proteins in this band and the metronidazole-sensitive phenotype.     

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.     

Kinetic analysis of reactive oxygen species generated by the in vitro reconstituted NADPH oxidase and xanthine oxidase systems

The nicotinamide adenine dinucleotide (NADH)/nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and the xanthine oxidase (XOD) systems generate reactive oxygen species (ROS). In the present study, to characterize the difference between the two systems, the kinetics of ROS generated by both the NADH oxidase and XOD systems were analysed by an electron spin resonance (ESR) spin trapping method using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), 5-(diethoxyphosphoryl)-5-methyl-pyrroline N-oxide (DEPMPO) and 5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO). As a result, two major differences in ROS kinetics were found between the two systems: (i) the kinetics of (?)OH and (ii) the kinetics of hydrogen peroxide. In the NADH oxidase system, the interaction of hydrogen peroxide with each component of the enzyme system (NADPH, NADH oxidase and FAD) was found to generate (?)OH. In contrast, (?)OH generation was found to be independent of hydrogen peroxide in the XOD system. In addition, the hydrogen peroxide level in the NADPH-NADH oxidase system was much lower than measured in the XOD system. This lower level of free hydrogen peroxide is most likely due to the interaction between hydrogen peroxide and NADPH, because the hydrogen peroxide level was reduced by ~90% in the presence of NADPH.     

Enzymatic activities in cell fractions of mycoplasmalike organisms purified from aster yellows-infected plants.

Mycoplasmalike organisms (MLOs), purified from aster yellows-infected plants were osmotically lysed, and the membranes were separated from the cytoplasmic fraction through differential centrifugation. Electron microscopic examinations of sections of the purified MLOs and the isolated membranes showed pleomorphic bodies and unit membranous empty vesicles, respectively. Cell fractions were tested for NADH oxidase, NADPH oxidase, ATPase, RNase, DNase, and p-nitrophenyl phosphatase activity. NADH oxidase and ATPase were confined to the membrane fraction and NADPH oxidase to the cytoplasmic fraction of the MLOs. para-Nitrophenyl phosphatase, RNase, and DNase activities were detected in both membrane and cytoplasmic fractions, but p-nitrophenyl phosphatase and RNase appeared to be associated with membranes and DNase with the cytoplasmic fraction. Glucose-6-phosphate dehydrogenase was found in the cytoplasmic fraction of the MLO cells. Our findings on the distribution of enzymes in MLO cells and cell fractions are the first basic documentation on nonhelical, nonculturable microbes parasitic to plants.     

Characterization of the mycoplasma membrane proteins. V. Release and localization of membrane-bound enzymes in Acholeplasma laidlawii.

The peripheral membrane protein fraction released by washing Acholeplasma laidlawii membranes with low-ionic strength buffers contained about 50% of the total membrane-bound ribonuclease and deoxyribonuclease activities. The ATPase, NADH oxidase and p-nitrophenylphosphatase activities remained bound to the membrane even when EDTA was added to the wash fluids, and thus appear to belong to the integral membrane protein group. Serving as a marker for peripheral membrane proteins, the membrane-bound ribonuclease activity was solubilized by bile salts much more effectively than the integral membrane-bound enzymes. On the other hand, the solubilized ribonuclease showed a much lower capacity to reaggregate with other solubilized membrane components to membranous structures. Yet, most of the ribonuclease molecules which were bound to the reaggregated membranes could not be released by low-ionic strength buffer. The reaggregated membranes differed from the native membranes in the absence of particles on their fracture faces obtained by freeze cleaving, and by their much higher labeling by the [125-I]lactoperoxidase iodination system. These results suggest that most of the proteins are exposed on the reaggregated membrane surfaces, with very little, if any, protein embedded in its lipid bilayer core. Enzyme disposition in the A. laidlawii membrane was studied by comparing the activity of isolated membranes with that of membranes of intact cells after treatment with pronase or with an antiserum to membranes. The data indicate the asymmetrical disposition of these activities, the ATPase and NADH oxidase being localized on the inner membrane surface, while the nucleases are exposed on the external membrane surface.     

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

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

Glutamate dehydrogenase from Mycoplasma laidlawii

Mycoplasma laidlawii possesses a single glutamate dehydrogenase (GDH) with dual coenzyme specificity [specificity for nicotinamide adenine dinucleotide (H) and nicotinamide adenine dinucleotide phosphate (H)]. A purification procedure is reported which results in an enzyme preparation with a specific activity of 79.5 units/mg and which displays only one significant protein band after gel electrophoresis. This one band was determined, by activity staining, to have all of the GDH nucleotide specificities. The molecular weight of the enzyme is 250,000 +/- 10%, and it has a subunit size of about 48,000. The enzyme exhibits measurable activity with aspartate and pyruvate but is inactive with eight other possible substrates. Purine nucleotides do not affect the activity. The K(m) for reduced nicotinamide adenine dinucleotide was 1.8 x 10(-4)m. The optimal substrate concentrations and pH optimum for each of the respective GDH activities are also reported.     

Formation of Hydrogen Peroxide by Group N Streptococci and Its Effect on Their Growth and Metabolism

The formation of hydrogen peroxide by group N streptococci was found to occur through the action of a reduced nicotinamide adenine dinucleotide (NADH) oxidase which catalyzed the oxidation of NADH by molecular oxygen. The enzyme was activated by flavine adenine dinucleotide. Whereas some of the hydrogen peroxide formed was removed through the action of an NADH peroxidase, sufficient accumulated in media to inhibit the growth, respiration, and viability of these organisms. The amount of hydrogen peroxide which accumulated varied among strains, and this variation could be related to differences in the properties of the NADH oxidase present.     

Physical Aggregation and Functional Reconstitution of Solubilized Membranes of Bacillus stearothermophilus

Isolated membranes of Bacillus stearothermophilus 2184D can be disrupted by treatment with sodium dodecyl sulfate (SDS). This disruption is attended by a decreased turbidity of membrane suspensions and a differential loss of activities of the electron transport system. Reduced methyl viologen (MVH)-nitrate reductase activity is insensitive to SDS treatment, whereas reduced nicotinamide adenine dinucleotide (NADH)-nitrate reductase and cyanide-sensitive NADH oxidase activities are decreased by 80% at an SDS concentration of 0.5 mg/mg of membrane protein. NADH-menadione reductase activity is unaffected at this SDS concentration, but at higher detergent levels it also decreases in activity. The abilities of NADH to reduce and nitrate to oxidize the cytochrome components of the membrane were also decreased after SDS treatment. Dilution of solubilized membrane in buffer containing divalent cation results in formation of an aggregate with an increased turbidity and reconstituted NADH-nitrate reductase and cyanide-sensitive NADH oxidase activities. Of several cations tested, magnesium was the most effective, and the reconstitution process was pH-dependent with an optimum at pH 7.4. Intact and aggregated membranes had similar densities and cytochrome contents, and the sensitivity of NADH-nitrate reductase to several inhibitors was similar in intact and reconstituted membranes.     

RESPIRATION AND PROTEIN SYNTHESIS IN ESCHERICHIA COLI MEMBRANE-ENVELOPE FRAGMENTS : IV. Chemical and Cytological Characterization and Biosynthetic Capabilities of Fragments Obtained by Mild Procedures

Membrane-envelope fragments have been isolated from Escherichia coli by comparatively mild techniques. The use of DNAase, RNAase, detergents, sonication, lysozyme, and ethylenediaminetetraacetate were avoided in the belief that rather delicate, but metabolically important, associations may exist between the plasma membrane and various cytoplasmic components. The membrane-envelope fragments have been characterized in terms of their content of major chemical components as well as their electron microscope appearance. Fractions containing membrane-envelope fragments were found to possess appreciable DNA- and protein-synthesizing activities. The fragments were rich in membrane content as determined by reduced nicotinamide adenine dinucleotide (NADH) oxidase activity and deficient in soluble components as measured by NADH dehydrogenase activity. The particulate fraction obtained between 20,000 g and 105,000 g and usually considered a ribosomal fraction was rich in membrane content and had a relatively high capacity for DNA synthesis. Envelope fragments sedimenting at 20,000 g attained very high levels of incorporation of amino acids into protein.     

Reconstitution of Micrococcus lysodeikticus reduced nicotinamide adenine dinucleotide and L-malate dehydrogenases with dehydrogenase-depleted membrane residues: a basis for restoration of oxidase activities

Deoxycholate disruption of Micrococcus lysodeikticus protoplast membranes resulted in solubilization of both l-malate and reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase enzymes (substrate: 2,6-dichlorophenolindophenol oxidoreductases). Insoluble residues contained cytochromes of the b, c, and a type. Solubilized dehydrogenases were reconstituted with insoluble residues by treatment of disrupted membranes with magnesium ions. Most of the solubilized l-malate and NADH dehydrogenase activities were precipitated by magnesium ions independent of enzyme reconstitution with insoluble residues. Reconstituted dehydrogenases explained the mechanism for restoration of disrupted l-malate and NADH oxidase activities (4). Black light irradiation inhibited oxidase activities of both native and reconstituted membranes. These irradiated membrane oxidases were partially restored by exogenous napthoquinones [K(2(20)) and K(2(50))] but not by CoQ((6)). Reconstitution experiments showed that native membrane napthoquinone was retained in the insoluble residues of deoxycholate-disrupted membranes.     

RESPIRATION AND PROTEIN SYNTHESIS IN ESCHERICHIA COLI MEMBRANE-ENVELOPE FRAGMENTS : I. Oxidative Activities with Soluble Substrates

This paper describes experiments conducted with membranous and soluble fractions obtained from Escherichia coli that had been grown on succinate, malate, or enriched glucose media. Oxidase and dehydrogenase activities were studied with the following substrates: nicotinamide adenine dinucleotide, reduced form (NADH), nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), succinate, malate, isocitrate, glutamate, pyruvate, and α-ketoglutarate. Respiration was virtually insensitive to poisons that are commonly used to inhibit mitochondrial systems, namely, rotenone, antimycin, and azide. Succinate dehydrogenase and NADH, NADPH, and succinate oxidases were primarily membrane-bound whereas malate, isocitrate, and NADH dehydrogenases were predominantly soluble. It was observed that E. coli malate dehydrogenase could be assayed with the dye 2,6-dichlorophenol indophenol, but that porcine malate dehydrogenase activity could not be assayed, even in the presence of E. coli extracts. The characteristics of E. coli NADH dehydrogenase were shown to be markedly different from those of a mammalian enzyme. The enzyme activities for oxidation of Krebs cycle intermediates (malate, succinate, isocitrate) did not appear to be under coordinate genetic control.