Flavensomycin, an inhibitor of enzyme reactions involving hydrogen transfer

The antifungal antibiotic flavensomycin inhibited the oxidation of amino acids and of glucose by Penicillium oxalicum. The compound inhibited l-amino acid oxidase (EC 1.4.3.2) activity for l-leucine and l-phenylalanine, and also d-amino acid oxidase (EC 1.4.3.3) in the oxidation for dl-alanine. The addition of flavin adenine dinucleotide, which is a cofactor for this enzyme, antagonized the action of the antibiotic. Glucose oxidase (EC 1.1.3.4) was also inhibited. The antibiotic inhibited the reduced nicotinamide adenine dinucleotide (NADH(2)) cytochrome c reductase (EC 1.6.2.1) as well as the much slower nonenzymatic reduction of this cytochrome by the nucleotide. Reduced cytochrome c was also oxidized nonenzymatically by flavensomycin. The antibiotic completely inhibited the action of rabbit muscle lactic dehydrogenase (EC 1.1.1.27) in promoting the reduction of pyruvate by NADH(2) but only slightly affected the reverse reaction. Alcohol dehydrogenase (EC 1.1.1.1) was also similarly inhibited. Flavensomycin prevented the reduction of nicotinamide adenine dinucleotide phosphate by isocitrate in the presence of isocitrate dehydrogenase (EC 1.1.1.42). The hexokinase (EC 2.7.1.1)-catalyzed phosphorylation of glucose, in which the adenosine triphosphate acts as a phosphate donor, was only slightly affected. Flavensomycin also inhibited the action of yeast lactate dehydrogenase (EC 1.1.2.3) on the reduction of cytochrome c. High concentrations of cytochrome c were antagonistic to this reaction. The results point to an interference with enzymatically controlled hydrogen or electron transfer as the mechanism of the antifungal activity of flavensomycin.     

Nitrate reductase complex of Escherichia coli K-12: participation of specific formate dehydrogenase and cytochrome b1 components in nitrate reduction

The participation of distinct formate dehydrogenases and cytochrome components in nitrate reduction by Escherichia coli was studied. The formate dehydrogenase activity present in extracts prepared from nitrate-induced cells of strain HfrH was active with various electron acceptors, including methylene blue, phenazine methosulfate, and benzyl viologen. Certain mutants which are unable to reduce nitrate had low or undetectable levels of formate dehydrogenase activity assayed with methylene blue or phenazine methosulfate as electron acceptor. Of nine such mutants, five produced gas when grown anaerobically without nitrate and possessed a benzyl viologen-linked formate dehydrogenase activity, suggesting that distinct formate dehydrogenases participate in the nitrate reductase and formic hydrogenlyase systems. The other four mutants formed little gas when grown anaerobically in the absence of nitrate and lacked the benzyl viologen-linked formate dehydrogenase as well as the methylene blue or phenazine methosulfate-linked activity. The cytochrome b(1) present in nitrate-induced cells was distinguished by its spectral properties and its genetic control from the major cytochrome b(1) components of aerobic cells and of cells grown anaerobically in the absence of nitrate. The nitrate-specific cytochrome b(1) was completely and rapidly reduced by 1 mm formate but was not reduced by 1 mm reduced nicotinamide adenine dinucleotide; ascorbate reduced only part of the cytochrome b(1) which was reduced by formate. When nitrate was added, the formate-reduced cytochrome b(1) was oxidized with biphasic kinetics, but the ascorbate-reduced cytochrome b(1) was oxidized with monophasic kinetics. The inhibitory effects of n-heptyl hydroxyquinoline-N-oxide on the oxidation of cytochrome b(1) by nitrate provided evidence that the nitrate-specific cytochrome is composed of two components which have different redox potentials but identical spectral properties. We conclude from these studies that nitrate reduction in E. coli is mediated by the sequential operation of a specific formate dehydrogenase, two specific cytochrome b(1) components, and nitrate reductase.     

Factor 420-dependent pyridine nucleotide-linked formate metabolism of Methanobacterium ruminantium.

Methanobacterium ruminantium was shown to possess a formate dehydrogenase which is linked to factor 420 (F420) as the first low-molecular-weight or anionic electron transfer coenzyme. Reduced F420 obtained from the formate dehydrogenase can be further linked to the formation of hydrogen via the previously described F420-dependent hydrogenase reaction, thus constituting an apparently simple formate hydrogenlyase system, or to the reduction of nicotinamide adenine dinucleotide phosphate via F420:nicotinamide adenine dinucleotide phosphate oxidoreductase. The results indicate that hydrogen and formate, the only known energy sources for M. ruminantium and many other methanogenic bacteria, should be essentially equivalent as sources of electrons in the metabolism of this organism.     

Formate dehydrogenase from Clostridium acidiurici

Partial purification of formate dehydrogenase from Clostridium acidiurici has been accomplished, and some properties of the enzyme have been determined. The molecular weight of the protein is at least 200,000 daltons. The enzyme showed marked instability to freezing and thawing and was inhibited strongly by oxygen and by light. Such inhibition was not reversed by incubation in the presence of thiol compounds. Cyanide inhibited the enzyme 90% at 0.1 mm concentrations, but ethylenediaminetetraacetate produced only slight inhibition at concentrations as high as 50 mm. The purified enzyme showed no ferredoxin activity in the Clostridium pasteurianum clastic system during pyruvate oxidation. Crude preparations of the enzyme could be coupled through ferredoxin to the reduction of nicotinamide adenine dinucleotide during formate oxidation, but the purified enzyme could not catalyze the reduction of pyridine nucleotides by formate in the presence of ferredoxin. Formate oxidation with the purified enzyme was readily coupled to benzyl viologen reduction, in which case ferredoxin was not required. An exchange between formate and bicarbonate was catalyzed by both crude and purified preparations of the enzyme, but the net synthesis of formate from CO(2) was not accomplished.     

Membrane-bound respiratory of Spirillum itersonii.

The membrane-bound respiratory system of the gram-negative bacterium Spirillum itersonii was investigated. It contains cytochromes b (558), c (550), and o (558) and beta-dihydro-nicotinamide adenine dinucleotide (NADH) and succinate oxidase activities under all growth conditions. It is also capable of producing D-lactate and alpha-glycerophosphate dehydrogenases when grown with lactate or glycerol as sole carbon source. Membrane-bound malate dehydrogenase was not detectable under any conditions, although there is high activity of soluble nicotinamide adenine dinucleotide: malate dehydrogenase. When grown with oxygen as the sole terminal electron acceptor, approximately 60% of the total b-type cytochrome is present as cytochrome o, whereas only 40% is present as cytochrome o in cells grown with nitrate in the presence of oxygen. Both NADH and succinate oxidase are inhibited by azide, cyanide, antimycin A, and 2-n-heptyl-4-hydroxyquinoline-N-oxidase at low concentrations. The ability of these inhibitors to completely inhibit oxidase activity at low concentrations and their effects upon the aerobic steady-state reduction levels of b- and c-type cytochromes as well as the aerobic steady-state reduction levels obtained with NADH, succinate, and ascorbate-dichlorophenolindophenol suggest that presence of an unbranched respiratory chain in S. itersonii with the order ubiquinone leads to b leads to c leads to c leads to oxygen.     

Properties of Nicotinamide Adenine Dinucleotide Phosphate-Dependent Formate Dehydrogenase from Clostridium thermoaceticum

Li, Lan-Fun (Western Reserve University School of Medicine, Cleveland, Ohio), Lars Ljungdahl, and Harland G. Wood. Properties of nicotinamide adenine dinucleotide phosphate-dependent formate dehydrogenase from Clostridium thermoaceticum. J. Bacteriol. 92: 405-412. 1966.-A nicotinamide adenine dinucleotide phosphate (NADP)-dependent formate dehydrogenase has been isolated from C. thermoaceticum. The enzyme is very sensitive to oxygen and requires sulfhydryl compounds for activity. The apparent K(m) at 50 C and pH 7.0 for NADP is 5.9 x 10(-5)m and for formate, 2.2 x 10(-4)m. The enzyme is most active at about 60 C and at pH values between 7.0 and 9.0. The enzyme catalyzes an exchange between C(14)O(2) and formate, which requires NADP, but net synthesis of formate from CO(2) and reduced nicotinamide adenine dinucleotide phosphate could not be demonstrated. The reaction does not involve ferredoxin.     

Spectrophotometric and Kinetic Study of Nitrite and Formate Oxidation in Nitrobacter winogradskyi

The reduction levels of cytochrome c and a(1) in intact Nitrobacter cells and cell-free extracts, during and after nitrite or formate oxidation, were examined in combination with the amperometric measurement of oxygen uptake. Quite different reduction patterns were observed when comparing nitrite oxidation by intact cells and cell-free extracts. An inverse relationship was observed between the rate of electron flow and the steady-state reduction level of cytochrome a(1). Parallel observations on nitrite oxidation, by use of formate and reduced nicotinamide adenine dinucleotide as electron donors, showed the influence of the high oxidation-reduction potential of the nitrite-nitrate system on cytochrome reduction. A value for the apparent activation energy of the overall nitrite oxidation process, amounting to 15 kcal, was found in a study of the temperature dependence of cytochrome reduction.     

A salt-inhibited cytochrome c reductase obtained from the moderately halophilic bacterium, Micrococcus halodenitrificans

A membrane-bound cytochrome c reductase from Micrococcus halodenitrificans was inhibited by NaCl at concentrations as low as 0.05 m. The inhibition was competitive with respect to cytochrome c and noncompetitive with respect to reduced nicotinamide adenine dinucleotide. Thus the effect of NaCl was to increase the apparent K(m) of the enzyme for cytochrome c and not to inhibit the intrinsic activity of the enzyme.     

Purification and Characterization of the Pseudomonas multivorans Glucose-6-Phosphate Dehydrogenase Active with Nicotinamide Adenine Dinucleotide

The Pseudomonas multivorans glucose-6-phosphate dehydrogenase (EC 1.1.1.49) active with nicotinamide adenine dinucleotide, which is inhibitable by adenosine-5'-triphosphate, was purified approximately 1,000-fold from extracts of glucose-grown bacteria, and characterized with respect to subunit composition, response to different inhibitory ligands, and certain other properties. The enzyme was found to be an oligomer composed of four subunits of about 60,000 molecular weight. Reduced nicotinamide adenine dinucleotide phosphate, but not reduced nicotinamide adenine dinucleotide, was found to be a potent inhibitor of its activity. The range of concentrations of reduced nicotinamide adenine dinucleotide phosphate over which inhibition occurred was about 100-fold lower than that for adenosine-5'-triphosphate. The data suggest that reduced nicotinamide adenine dinucleotide phosphate may play an important role in regulation of hexose phosphate metabolism in P. multivorans. Antisera prepared against the purified enzyme strongly inhibited its activity, but failed to inhibit the activity of the nicotinamide adenine dinucleotide phosphate-specific glucose-6-phosphate dehydrogenase which is also present in extracts of this bacterium. Immunodiffusion experiments confirmed the results of the enzyme inhibition studies, and failed to support the idea that the two glucose-6-phosphate dehydrogenase species from P. multivorans represent different oligomeric forms of the same protein.     

Nicotinamide Adenine Dinucleotide Phosphate-Dependent Formate Dehydrogenase from Clostridium thermoaceticum: Purification and Properties

The nicotinamide adenine dinucleotide phosphate (NADP)-dependent formate dehydrogenase in Clostridium thermoaceticum used, in addition to its natural electron acceptor, methyl and benzyl viologen. The enzyme was purified to a specific activity of 34 (micromoles per minute per milligram of protein) with NADP as electron acceptor. Disc gel electrophoresis of the purified enzyme yielded two major and two minor protein bands, and during centrifugation in sucrose gradients two components of apparent molecular weights of 270,000 and 320,000 were obtained, both having formate dehydrogenase activity. The enzyme preparation catalyzed the reduction of riboflavine 5'-phosphate flavine adenine dinucleotide and methyl viologen by using reduced NADP as a source of electrons. It also had reduced NADP oxidase activity. The enzyme was strongly inhibited by cyanide and ethylenediaminetetraacetic acid. It was also inhibited by hypophosphite, an inhibition that was reversed by formate. Sulfite inhibited the activity with NADP but not with methyl viologen as acceptor. The apparent K(m) at 55 C and pH 7.5 for formate was 2.27 x 10(-4) M with NADP and 0.83 x 10(-4) with methyl viologen as acceptor. The apparent K(m) for NADP was 1.09 x 10(-4) M and for methyl viologen was 2.35 x 10(-3) M. NADP showed substrate inhibition at 5 x 10(-3) M and higher concentrations. With NADP as electron acceptor, the enzyme had a broad pH optimum between 7 and 9.5. The apparent temperature optimum was 85 C. In the absence of substrates, the enzyme was stable at 70 C but was rapidly inactivated at temperatures above 73 C. The enzyme was very sensitive to oxygen but was stabilized by thiol-iron complexes and formate.     

Use of tetrazolium salts for electron transport studies in meningopneumonitis. I. Reduced nicotinamide adenine dinucleotide system

Allen, Emma G. (Downstate Medical Center, Brooklyn, N.Y.). Use of tetrazolium salts for electron transport studies in meningopneumonitis. I. Reduced nicotinamide adenine dinucleotide system. J. Bacteriol. 90:1505-1512. 1965.-Purified preparations of meningopneumonitis virus (MP) prepared from allantoic fluids of infected chick embryo reduce several tetrazolium salts in the presence of reduced nicotinamide adenine dinucleotide under both aerobic and anaerobic conditions. The pattern of reduction by MP differs from that seen in normal allantoic membrane homogenates, and is inhibited by several cations but not by KCN, atabrine, amytal, antimycin A, or 2,3-dimercaptopropanol (BAL). The reduction of cytochrome c by purified preparations of MP differs from its reduction of tetrazolium salts in that the cytochrome reaction is completely inhibited by BAL and partially inhibited by amytal, atabrine, and antimycin A. The cytochrome reductase of normal allantoic membrane preparations is completely inhibited by each of these compounds.     

Purification and Characterization of the Two 6-Phosphogluconate Dehydrogenase Species from Pseudomonas multivorans

The two species of 6-phosphogluconate dehydrogenase (EC 1.1.1.43) from Pseudomonas multivorans were resolved from extracts of gluconate-grown bacteria and purified to homogeneity. Each enzyme comprised between 0.1 and 0.2% of the total cellular protein. Separation of the two enzymes, one which is specific for nicotinamide adenine dinucleotide phosphate and the other which is active with nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate was facilitated by the marked difference in their respective isoelectric points, which were at pH 5.0 and 6.9. Comparison of the subunit compositions of the two enzymes indicated that they do not share common peptide chains. The enzyme active with nicotinamide adenine dinucleotide was composed of two subunits of about 40,000 molecular weight, and the nicotinamide adenine dinucleotide phosphate-specific enzyme was composed of two subunits of about 60,000 molecular weight. Immunological studies indicated that the two enzymes do not share common antigenic determinants. Reduced nicotinamide adenine dinucleotide phosphate strongly inhibited the 6-phosphogluconate dehydrogenase active with nicotinamide adenine dinucleotide by decreasing its affinity for 6-phosphogluconate. Guanosine-5'-triphosphate had a similar influence on the nicotinamide adenine dinucleotide phosphate-specific 6-phosphogluconate dehydrogenase. These results in conjunction with other data indicating that reduced nicotinamide adenine dinucleotide phosphate stimulates the conversion of 6-phosphogluconate to pyruvate by crude bacterial extracts suggest that in P. multivorans, the relative distribution of 6-phosphogluconate into the pentose phosphate and Entner-Doudoroff pathways might be determined by the intracellular concentrations of reduced nicotinamide adenine dinucleotide phosphate and purine nucleotides.     

Enzymatic Synthesis of l-Carnitine by Reduction of an Achiral Precursor: the Problem of Reduced Nicotinamide Adenine Dinucleotide Recycling

Synthesis of l-carnitine has been carried out by the enzymatic reduction of the carbonyl group of the achiral precursor 3-dehydrocarnitine with the oxidized nicotinamide adenine dinucleotide-linked carnitine dehydrogenase. Various enzymatic or chemical systems have been tested to regenerate the reduced nicotinamide adenine dinucleotide oxidized in the reduction of 3-dehydrocarnitine. Because of the instability of this compound in aqueous solutions, it was added by continuous feeding as a rate-limiting constituent in the reaction mixture. Under these conditions, conversion yields of 95% were achieved with the glucose plus glucose dehydrogenase system. A total number of 530 reduced nicotinamide adenine dinucleotide recyclings was obtained with this system for a production of 45 g of l-carnitine per liter. The stabilities of the oxidized nicotinamide adenine dinucleotide and the reduced nicotinamide adenine dinucleotide have been determined at various pH values. In view of these results, several possible strategies for enzymatic syntheses with the reduced nicotinamide adenine dinucleotide as a regenerable coenzyme are discussed.     

Mechanism of Action of the Antifungal Antibiotic Pyrrolnitrin

Pyrrolnitrin at 10 mug/ml inhibited the growth of Saccharomyces cerevisiae, Penicillium atrovenetum, and P. oxalicum. The primary site of action of pyrrolnitrin on S. cerevisiae was the terminal electron transport system between succinate or reduced nicotinamide adenine dinucleotide (NADH) and coenzyme Q. At growth inhibitory concentrations, pyrrolnitrin inhibited endogenous and exogenous respiration immediately after its addition to the system. In mitochondrial preparations, the antibiotic inhibited succinate oxidase, NADH oxidase, succinate-cytochrome c reductase, NADH-cytochrome c reductase, and succinate-coenzyme Q(6) reductase. In addition, pyrrolnitrin inhibited the antimycin-insensitive reduction of dichlorophenolindophenol and of the tetrazolium dye 2,2'-di-p-nitrophenyl-(3,3'-dimethoxy-4,4'-bi-phenylene)5,5'-diphenylditetrazolium. The reduction of another tetrazolium dye, 2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium chloride, that was antimycin-sensitive, was also inhibited by pyrrolnitrin. The antibiotic had no effect on the activity of cytochrome oxidase, and it did not appear to bind with flavine adenine dinucleotide, the coenzyme of succinic dehydrogenase. In whole cells of S. cerevisiae, pyrrolnitrin inhibited the incorporation of (14)C-glucose into nucleic acids and proteins. It also inhibited the incorporation of (14)C-uracil, (3)H-thymidine, and (14)C-amino acids into ribonucleic acid, deoxyribonucleic acid, and protein, respectively. The in vitro protein synthesis in Rhizoctonia solani and Escherichia coli was not affected by pyrrolnitrin. Pyrrolnitrin also inhibited the uptake of radioactive tracers, but there was no general damage to the cell membranes that would result in an increased leakage of cell metabolites. Apparently, pyrrolnitrin inhibits fungal growth by inhibiting the respiratory electron transport system.     

Respiratory systems and cytochromes in Campylobacter fetus subsp. intestinalis.

Cell suspensions of Campylobacter fetus subsp. intestinalis grown microaerophilically in complex media consumed oxygen in the presence of formate, succinate, and DL-lactate, and membranes had the corresponding dehydrogenase activities. The cells and membranes also had ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine oxidase activity which was cyanide sensitive. The fumarate reductase activity in the membranes was inhibited by p-chloromercuriphenylsulfonate, and this enzyme was probably responsible for the succinate dehydrogenase activity. Cytochrome c was predominant in the membranes, and a major proportion of this pigment exhibited a carbon monoxide-binding spectrum. Approximately 60% of the total membrane cytochrome c, measured with dithionite as the reductant, was also reduced by ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine. A similar proportion of the membrane cytochrome c was reduced by succinate under anaerobic conditions, whereas formate reduced more than 90% of the total cytochrome under these conditions. 2-Heptyl-4-hydroxyquinoline-N-oxide inhibited reduction of cytochrome c with succinate, and the reduced spectrum of cytochrome b became evident. The inhibitor delayed reduction of cytochrome c with formate, but the final level of reduction was unaffected. We conclude that the respiratory chain includes low- and high-potential forms of cytochromes c and b; the carbon monoxide-binding form of cytochrome c might function as a terminal oxidase.     

Reduction of iron and synthesis of protoheme by Spirillum itersonii and other organisms.

Membranes from Spirillum itersonii reduce ferric iron to ferrous iron with reduced nicotinamide adenine dinucleotide or succinate as a source of reductant. Iron reduction was measured spectrophotometrically at 562 nm using ferrozine, which chelates ferrous iron specifically. Reduced nicotinamide adenine dinucleotide or succinate was also effective as a source of iron. The effects of respiratory inhibitors suggested that reduction of iron occurs at one or more sites on the respiratory chain before cytochrome c. Reduction of iron and synthesis of protoheme with the physiological reductants were also observed with crude extracts of other bacteria, including Rhodopseudomonas spheroides, Rhodopseudomonas capsulata, Paracoccus denitrificans, and Escherichia coli. The effect of oxygen upon reduction of iron and formation of protoheme was examined with membranes from S. itersonii, using succinate as a source of reductant. Both systems were inhibited by oxygen, but this effect was completely reversed by addition of antimycin A. We conclude that reduced components of the respiratory chain serve as reductants for ferric iron, but with oxygen present they are oxidized preferentially by the successive members of the chain. This could be a mechanism for regulating synthesis of heme and cytochrome by oxygen.     

The ultrastructural cytochemistry of lactic dehydrogenase, succinic dehydrogenase, dihydro-nicotinamide adenine dinucleotide diaphorase and cytochrome oxidase activities in hair cell mitochondria of the guinea pig cochlea.

The use of cinnamyl nitroblue tetrazolium chloride (DS-NBT) in dehydrogenase experiments (lactic dehydrogenase, succinic dehydrogenase, nicotinamide adenine dinucleotide diaphorase) and 3,3'-diaminobenzidine tetrahydrochloride (DAB) in cytochrome oxidase experiments indicated that mitochondrial oxidoreduction reactions from nicotinamide adenine dinucleotide to cytochrome oxidase are located on the inner mitochondrial membrane in the outer compartment and the intracristate spaces. These reactions behave according to the chemiosmotic hypothesis. The cochlear hair cell mitochondria are cytochemically indistinguishable from free liver mitochondria. The heterogeneous mitochondrial staining pattern is related to the osmolarity of the incubation media, solubility of the enzymes and pH of the medium, but not to the fixation method.     

Nicotinamide Adenine Dinucleotide Phosphate-specific Isocitrate Dehydrogenase from a Higher Plant: Isolation and Characterization 1

Nicotinamide adenine dinucleotide phosphate-specific isocitrate dehydrogenase was extracted from etiolated pea (Pisum sativum L.) seedlings and was purified 65-fold. The purified enzyme exhibits one predominant protein band by polyacrylamide gel electrophoresis, which corresponds to the dehydrogenase activity as measured by the nitro blue tetrazolium technique. The reaction is readily reversible, the pH optima for the forward (nicotinamide adenine dinucleotide phosphate reduction) and reverse reactions being 8.4 and 6.0, respectively. The enzyme has different cofactor and inhibitor characteristics in the two directions. Manganese ions can be used as a cofactor for the reaction in each direction but magnesium ions only act as a cofactor in the forward reaction. Zinc ions, and to a lesser extent calcium ions, inhibit the enzyme at low concentrations when magnesium but not manganese is the metal activator. It is suggested that there is a fundamental difference between magnesium and manganese in the activation of the enzyme. The enzyme shows normal kinetics and the Michaelis contant for each substrate was determined. The inhibition by nucleotides, nucleosides, reaction products, and related compounds was studied. The enzyme shows a linear response to the mole fraction of reduced nicotinamide adenine dinucleotide phosphate when total nicotinamide adenine dinucleotide phosphate (nicotinamide adenine dinucleotide phosphate plus reduced nicotinamide adenine dinucleotide phosphate) is kept constant. Isocitrate in the presence of divalent metal ions will protect the enzyme from inactivation by p-chloromercuribenzoate. Protection is also afforded by manganese ions alone but not by magnesium ions alone There is a concerted inhibition of the enzyme by oxalacetate and glyoxylate.     

Hexuronic acid dehydrogenase of Agrobacterium tumefaciens

Growth of Agrobacterium tumefaciens on d-glucuronic acid (GlcUA) or d-galacturonic acid (GalUA) induces formation of hexuronic acid dehydrogenase [d-aldohexuronic acid: nicotinamide adenine dinucleotide (NAD) oxidoreductase]. The dehydrogenase, which irreversibly converts GlcUA or GalUA to the corresponding hexaric acid with the concomitant reduction of NAD, but not of nicotinamide adenine dinucleotide phosphate was purified 60-fold by MnCl(2) treatment, (NH(4))(2)SO(4) fractionation, chromatography on diethylaminoethyl Sephadex and negative adsorption with Ca(3)(PO(4))(2) gel. The pH optimum is 8.0. Other uronic acids, aldohexoses, aldopentoses, and polyols, are not substrates. Reduced nicotinamide adenine dinucleotide is an inhibitor strictly competitive with NAD. Kinetic data indicate that the dehydrogenase induced by growth on GlcUA may not be identical with that induced by growth on GalUA.     

Cytochrome p-450-dependent hydroxylation of lauric Acid at the subterminal position and oxidation of unsaturated analogs in wheat microsomes

Microsomes from etiolated wheat (Triticum aestivum L. cv Etoile de Choisy) shoots catalyzed the reduced nicotinamide adenine dinucleotide phosphate-dependent hydroxylation of lauric acid predominantly at the subterminal or (ω-1) position (65%). Minor amounts of 10-hydroxy- (31%) and 9-hydroxylaurate (4%) were also formed. The reaction was catalyzed by cytochrome P-450, since enzyme activity was strongly inhibited by tetcyclacis, carbon monoxide, and antibodies against NADPH-cytochrome c (P-450)-reductase. The apparent K_m for lauric acid was estimated to be 8.5 ± 2.0 μm. Seed treatment with the safener naphthalic acid anhydride or treatment of seedlings with phenobarbital increased cytochrome P-450 content and lauric acid hydroxylase (LAH) activity of the microsomes. A combination of both treatments further stimulated LAH activity. A series of radiolabeled unsaturated lauric acid analogs (8-, 9-, 10-, and 11-dodecenoic acids) was used to explore the regioselectivity and catalytic capabilities of induced wheat microsomes. It has been found that wheat microsomes catalyzed the reduced nicotinamide adenine dinucleotide phosphate-dependent epoxidation of sp2 carbons concurrently with hydroxylation at saturated positions. The regioselectivity of oxidation of the unsaturated substrates and that of lauric acid were similar. Preincubation of wheat microsomes with reduced nicotinamide adenine dinucleotide phosphate and 11-dodecenoic acid resulted in a partial loss of LAH activity.