Diaphorase Reactions Research Articles

Use of a Reverse Micelle System for Study of Oligomeric Structure of NAD+-Reducing Hydrogenase from Ralstonia eutropha H16

Inclusion of an oligomeric enzyme, NAD+-dependent hydrogenase from the hydrogen-oxidizing bacterium Ralstonia eutropha, into a system of reverse micelles of different sizes resulted in its dissociation into catalytically active heterodimers and subunits, which were characterized in reactions with various substrates. It was found that: 1) the native tetrameric form of this enzyme catalyzes all types of studied reactions; 2) hydrogenase dimer, HoxHY, is a minimal structural unit catalyzing hydrogenase reaction with an artificial electron donor, reduced methyl viologen; 3) all structural fragments containing FMN and NAD+/NADH-binding sites exhibit catalytic activity in diaphorase reactions with one- and two-electron acceptors; 4) small subunits, HoxY and HoxU also exhibit activity in diaphorase reactions with artificial acceptors. These results can be considered as indirect evidence that the second FMN molecule may be associated with one of the small subunits (HoxY or HoxU) of the hydrogenase from R. eutropha.

Inhibition of pea ferredoxin-NADP(H) reductase by Zn-ferrocyanide.

Ferredoxin-NADP(H) reductases (FNRs) represent a prototype of enzymes involved in numerous metabolic pathways. We found that pea FNR ferricyanide diaphorase activity was inhibited by Zn2+ (Ki 1.57 microM). Dichlorophenolindophenol diaphorase activity was also inhibited by Zn2+ (Ki 1.80 microM), but the addition of ferrocyanide was required, indicating that the inhibitor is an arrangement of both ions. Escherichia coli FNR was also inhibited by Zn-ferrocyanide, suggesting that inhibition is a consequence of common structural features of these flavoenzymes. The inhibitor behaves in a noncompetitive manner for NADPH and for artificial electron acceptors. Analysis of the oxidation state of the flavin during catalysis in the presence of the inhibitor suggests that the electron-transfer process between NADPH and the flavin is not significantly altered, and that the transfer between the flavin and the second substrate is mainly affected. Zn-ferrocyanide interacts with the reductase, probably increasing the accessibility of the prosthetic group to the solvent. Ferredoxin reduction was also inhibited by Zn-ferrocyanide in a noncompetitive manner, but the observed Ki was about nine times higher than those for the diaphorase reactions. The electron transfer to Anabaena flavodoxin was not affected by Zn-ferrocyanide. Binding of the apoflavodoxin to the reductase was sufficient to overcome the inhibition by Zn-ferrocyanide, suggesting that the interaction of FNRs with their proteinaceous electron partners may induce a conformational change in the reductase that alters or completely prevents the inhibitory effect.

Catalysis of diaphorase reactions by Mycobacterium tuberculosis lipoamide dehydrogenase occurs at the EH4 level.

Lipoamide dehydrogenase catalyzes the reversible NAD(+)-dependent oxidation of the dihydrolipoyl cofactors that are covalently attached to the acyltransferase components of the pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, and glycine reductase multienzyme complexes. It contains two redox centers: a tightly, but noncovalently, bound FAD and an enzymic disulfide, each of which can accommodate two electrons. In the two-electron-reduced enzyme (EH(2)), the disulfide is reduced while the FAD cofactor is oxidized. In the four-electron-reduced enzyme (EH(4)), both redox centers are reduced. Lipoamide dehydrogenase can also catalyze the NADH-dependent reduction of alternative electron acceptors such as 2,6-dichlorophenolindophenol, ferricyanide, quinones, and molecular oxygen (O(2)). To determine the mechanism of these "diaphorase" reactions, we generated the EH(2) and EH(4) forms of Mycobacterium tuberculosis lipoamide dehydrogenase and rapidly mixed these enzyme forms with d,l-lipoylpentanoate, 2,6-dimethyl-1,4-benzoquinone, and O(2), in a stopped-flow spectrophotometer at pH 7.5 and 4 degrees C. EH(2) reduced d,l-lipoylpentanoate >/=100 times faster than EH(4) did. Conversely, EH(4) reduced 2,6-dimethyl-1,4-benzoquinone and molecular oxygen 90 and 40 times faster than EH(2), respectively. Comparison of the rates of reduction of the above substrates by EH(2) and EH(4) with their corresponding steady-state kinetic parameters for kinetic competence leads to the conclusion that reduction of lipoyl substrates occurs with EH(2) while reduction of diaphorase substrates occurs with EH(4).

Progressive reorganization of the myenteric plexus during one year following reanastomosis of the ileum of the guinea pig

The enteric nervous system appears to play a pivotal role in the functional recovery of the gastrointestinal tract after partial resection and reanastomosis, but the structural changes following surgery are not fully understood. The present study was designed to clarify the processes of myenteric plexus regeneration up to one year after transection and reanastomosis of the ileum of the guinea pig. The following techniques were used: nicotinamide adenine dinucleotide (NADH) diaphorase histochemistry, immunostaining of neuron-specific enolase (NSE) in whole-mount preparations, and transmission electron microscopy. Two months after transection and reanastomosis, myenteric ganglion cells with NADH diaphorase reactions were scarce in the center of the lesion, and were less numerous in adjacent areas (3 mm in width) than in the control ileum. In the areas adjacent to the lesion, a few large extraganglionic neurons that did not completely compensate for the loss of ganglion neurons were observed. The remaining ileum showed no changes in NADH diaphorase staining pattern at this stage. Two to 12 months after transection and reanastomosis, ectopic large neurons gradually increased in number not only in the areas adjacent to the lesion but also in part of the remaining ileum, up to 10 cm from the lesion. Concomitantly, large ganglion neurons decreased in number in these areas. In other ileal regions (more than 10 cm distant from the site of transection), no obvious changes in NADH diaphorase staining were noted throughout the observation period. The outgrowth of NSE-containing nerve fibers from the severed stumps was seen two weeks after transection. Six weeks later, numerous bundles of fine nerve fibers with NSE were shown to interconnect the oral and anal cut ends of the myenteric plexus, but they exhibited no subsequent alterations. Transmission electron microscopy revealed that regenerating nerve fiber bundles appeared initially among irregularly arranged smooth muscle cells eight weeks after the operation, as expected from light-microscopic observations. These findings suggest that myenteric ganglion cell bodies, unlike myenteric nerve fibers, require a longer term of reconstruction than previously believed after transection and reanastomosis of the ileum of the guinea pig.

Interference of electron transport inhibitors with desaturation of monogalactosyl diacylglycerol in intact chloroplasts

Isolated intact chloroplasts are able to desaturate fatty acids in newly synthesized monogalactosyl diacylglycerol. By analogy with other systems, this desaturation might be expected to involve electron carriers. The effects of electron transport inhibitors on chloroplast lipid-linked desaturation were therefore investigated. Because desaturation occurs in the dark and is not inhibited by compounds specifically blocking photosystem II, it appeared that the photosystems themselves did not participate. Several compounds that prevent enzymatic reoxidation of plastoquinol in thylakoid membranes at the Q Z site or withdraw electrons from this lipophilic electron carrier inhibited desaturation in the dark. This inhibition could not be reversed by adding chemicals that donate electrons to photosystem I, indicating that carriers past the cytochrome b/f complex were not involved. Inhibitors of cyclic electron transport interfered with desaturation only at rather high concentrations or not at all. Additional compounds that block the reduction of quinones were slightly inhibitory. Dithioerythritol and KCN also inhibited desaturation, although their exact mode of action is unknown. Dinitrophenyl-iodonitrothymol (DNP-INT), stigmatellin, and myxothiazol did not block desaturation at concentrations that inhibited photosynthetic electron flow through the Q Z site very efficiently. Therefore, these results argue against an involvement of the Q Z site in desaturation. Accordingly, the inhibition by the other compounds seemingly interfering at the same site as well as that by electron acceptors could be due to interference at a different redox step in desaturation. In vitro these compounds function also as electron acceptors in diaphorase reactions catalyzed by ferredoxin:NADP oxidoreductase.

Multifunctional activities of yeast glutathione reductase

1. 1. Yeast glutathione reductase exists in a single molecular form which exhibits preferred NADPH and weak NADH linked multifunctional activities. 2. 2. Kinetic parameters for the NADPH and NADH linked reductase, transhydrogenase, electron transferase and diaphorase reactions have been determined. 3. 3. The functional preference for the NADPH linked reductase reaction is kinetically related to the high catalytic efficiency and low dissociation constants for substrates. 4. 4. NADP + and NAD + may interact with two different sites or different kinetic forms of the enzyme. 5. 5. The active site disulfide and histidine are required for the reductase activity but are not essential to the transhydrogenase, electron transferase and diaphorase activities. 6. 6. Amidation of carboxyl groups and Co(II) chelation of glutathione reductase facilitate the electron transferase reaction presumably by encouraging the formation of an anionic flavosemiquinone.

Superoxide anion involvement in NBT reduction catalyzed by nadph-cytochrome P-450 reductase: A pitfall

FEBS LettersVolume 89, Issue 1 p. 26-28 Full-length articleFree Access Superoxide anion involvement in NBT reduction catalyzed by nadph-cytochrome P-450 reductase: A pitfall Christian Auclair, Corresponding Author Christian Auclair U160 (INSERM), ERA 573 (CNRS), Université Paris VII and Hôpital Bichat, Paris, FranceC. Auclair, Laboratoire Central d'Immunologie et d'Hématologie, Hôpital Bichat, 170, bd Ney, 75877 Paris Cédex 18, FranceSearch for more papers by this authorMartine Torres, Martine Torres U160 (INSERM), ERA 573 (CNRS), Université Paris VII and Hôpital Bichat, Paris, FranceSearch for more papers by this authorJacques Hakim, Jacques Hakim U160 (INSERM), ERA 573 (CNRS), Université Paris VII and Hôpital Bichat, Paris, FranceSearch for more papers by this author Christian Auclair, Corresponding Author Christian Auclair U160 (INSERM), ERA 573 (CNRS), Université Paris VII and Hôpital Bichat, Paris, FranceC. Auclair, Laboratoire Central d'Immunologie et d'Hématologie, Hôpital Bichat, 170, bd Ney, 75877 Paris Cédex 18, FranceSearch for more papers by this authorMartine Torres, Martine Torres U160 (INSERM), ERA 573 (CNRS), Université Paris VII and Hôpital Bichat, Paris, FranceSearch for more papers by this authorJacques Hakim, Jacques Hakim U160 (INSERM), ERA 573 (CNRS), Université Paris VII and Hôpital Bichat, Paris, FranceSearch for more papers by this author First published: May 01, 1978 https://doi.org/10.1016/0014-5793(78)80514-6Citations: 77AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. References 1 I. Fridovich, W.A. Pryor Free radicals in Biology (1977), Academic Press New York 239– 277. 2 V. Massey, S. Strickland, S.G. Mayhew, L.G. Howell, P.G. Engel, R.G. Mathewo, M. Shuman, P.A. Sullivan, Biochem. Biophys. Res. Commun., 36, (1969), 891– 897. 3 W.H. Orme-Johnson, H. Beinert, Biochem. Biophys. Res. Commun., 36, (1969), 905– 911. 4 S.G. Slingar, J.D. Lipscomb, P.G. Debrunner, I.C. Gunsalus, Biochem. Biophys. Res. Commun., 61, (1974), 290– 296. 5 C. Beauchamp, I. Fridovich, Anal. Biochem., 44, (1971), 276– 287. 6 J.M. McCord, I. Fridovich, J. Biol. Chem., 244, (1969), 6049– 6055. 7 V. Mishin, A. Pokrovsky, V.V. Lyakhovich, Biochem. J., 154, (1976), 307– 310. 8 J.A. Knapp, J.D. Dignam, H.W. Strobel, J. Biol. Chem., 252, (1977), 437– 443. 9 P. Böhlen, S. Stein, W. Dairman, S. Udenfriend, Arch. Biochem. Biophys., 155, (1973), 213– 220. 10 C.H. Williams, P.D. Boyer The Enzymes 13, (1976), 165– 173. part C 11 J.L. Vermilion, M.J. Coon, Biochem. Biophys. Res. Commun., 60, (1974), 1315– 1322. Citing Literature Volume89, Issue1May 01, 1978Pages 26-28 ReferencesRelatedInformation

Trypsin inhibition of photosynthetic pyridine nucleotide reduction

1. 1. Photosynthetic pyridine nucleotide reduction is rapidly inhibited by trypsin, the extent of which depends on (i) the order of addition of components to reaction mixtures and (ii) the amount of trypsin added. The inhibited rate appears to be a steady-state rate and is a function of the concentration of trypsin. 2. 2. A partial restoration of the rapidly inhibited rate of NADP + reduction can be achieved by subsequent addition of trypsin inhibitor. 3. 3. Rapid inhibition by trypsin is not manifested in either methylviologen-catalyzed oxygen consumption or ferredoxin-dependent cytochrome c photoreduction. Nor is there any effect of trypsin on ferredoxin-NADP + reductase-catalyzed diaphorase reactions. 4. 4. It is suggested that trypsin binds rapidly to the chloroplast membrane and inhibits electron flow from the primary acceptor of Photosystem I to ferredoxin-NADP + reductase.

Purification and characterization of ferredoxin-nicotinamide adenine dinucleotide phosphate reductase from a nitrogen-fixing bacterium.

Evidence suggesting that Bacillus polymyxa has an active ferredoxin-NADP(+) reductase (EC 1.6.99.4) was obtained when NADPH was found to provide reducing power for the nitrogenase of this organism; direct evidence was provided when it was shown that B. polymyxa extracts could substitute for the native ferredoxin-NADP(+) reductase in the photochemical reduction of NADP(+) by blue-green algal particles. The ferredoxin-NADP(+) reductase was purified about 80-fold by a combination of high-speed centrifugation, ammonium sulfate fractionation, and chromatography on Sephadex G-100 and diethylaminoethyl-cellulose. The molecular weight was estimated by gel filtration to be 60,000. A small amount of the enzyme was further purified by polyacrylamide gel electrophoresis and shown to be a flavoprotein. The reductase was specific for NADPH in the ferredoxin-dependent reduction of cytochrome c and methyl viologen diaphorase reactions; furthermore, NADP(+) was the acceptor of preference when the electron donor was photoreduced ferredoxin. The reductase also has an irreversible NADPH-NAD(+) transhydrogenase (reduced-NADP:NAD oxidoreductase, EC 1.6.1.1) activity, the rate of which was proportional to the concentration of NAD (K(m) = 5.0 x 10(-3)M). The reductase catalyzed electron transfer from NADPH not only to B. polymyxa ferredoxin but also to the ferredoxins of Clostridium pasteurianum, Azotobacter vinelandii, and spinach chloroplasts, although less effectively. Rubredoxin from Clostridium acidi-urici and azotoflavin from A. vinelandii also accept electrons from the B. polymyxa reductase. The pH optima for the various reactions catalyzed by the B. polymyxa ferredoxin-NADP reductase are similar to those of the chloroplast reductase. NAD and acetyl-coenzyme A, which obligatorily activate NADPH- and NADH-ferredoxin reductases, respectively, in Clostridium kluyveri, have no effect on B. polymyxa reductase.

Histochemical localization of brain succinic semialdehyde dehydrogenase--a -aminobutyric acid degradative enzyme.

Succinic semialdehyde dehydrogenase is the final enzyme in the degradative pathway of γ-aminobutyric acid, a postulated inhibitory synaptic transmitter. This study reports a specific, sensitive technique for the localization of succinic semialdehyde dehydrogenase in rat brain. The technique utilizes the direct reduction of nitroblue tetrazolium by reduced pyridine nucleotide at an alkaline pH, a mechanism of formazan production substantiated by biochemical studies. The succinic semialdehyde dehydrogenase histochemical reaction is completely inhibited by p-hydroxybenzaldehyde and m-hydroxybenzaldehyde, compounds which have no effect on succinic acid dehydrogenase and reduced diphosphopyridine nucleotide diaphorase. Conversely, succinic acid dehydrogenase and reduced diphosphopyridine nucleotide diaphorase are inhibited by malonate and nordihydroguaiaretic acid, respectively, substances which have no effect on succinic semialdehyde dehydrogenase. The distribution and intensity of staining for succinic semialdehyde dehydrogenase are different from that seen following the succinic acid dehydrogenase and reduced diphosphopyridine nucleotide diaphorase reactions and can be localized to groups of neurons and their cell processes, e.g., the Purkinje cell dendritic tree, and in specific regions of neuropil.

Some histochemical and biochemical ovservations on the lymphatic tissues of highly inbred white leghorns.

AbstractLymphatic tissues of inbred lines of White Leghorn chickens and 20‐day embryos (inbreeding coefficient exceeds 95%) were used in the experiments, e.g. line 6 was susceptible, line 7 was resistant, and line 15 I was intermediate in response to the virus. Enzyme reactions were studied in cryostat‐cut sections of tissues and in tissue minces by colorimetric procedures. Numbers of isozymes and proteins of lymphatic tissues were resolved by disc gel electrophoresis.Colorimetric tests showed that intensities of lactate, malate, isocitrate and succinic dehydrogenase catalyzed reactions were higher in the bursae of 15 I 20‐day embryos than they were in bursae of either line 6 or 7 embryos. Intensity of dehydrogenase reactions of the spleen (15 I embryos) exceeded that found in line 6 and 7 embryos. Intensity of diaphorase reactions in the spleen and thymus was fairly uniform in all lines of embryos. Intensity of DPN diaphorase reactions in the bursae of line 15 I embryos exceeds that found in either line 6 or 7 embryos. Intensity of enzyme reactions leveled off to become fairly uniform in lymphatic tissues of chickens 3–4 weeks post hatching with the exception that dehydrogenase reactions were less intense in the thymus of 15 I chickens.Photodensitometer scans of acrylamide gel columns showed that proteins of line 6 lymphatic tissues combined with less Amido black 10B than lymphatic proteins of either line 15 I or 7 embryos. There was fairly good agreement between concentrations of strong mobility (components 1–9) and weak mobility (components 10–16) in lymphatic tissues of all lines of embryos with the exception that strong mobility proteins were about twice as concentrated in line 15 I bursae. Variable numbers of lactate isozymes were found in the lymphatic tissues of 20‐day embryos.

OXIDATIVE AND HYDROLYTIC ENZYMES OF HOMOZYGOUS DYSTROPHIC AND HETEROZYGOUS MUSCLE OF THE HOUSE MOUSE

Histochemical procedures were used for the identification of oxidative and hydrolytic enzymes of homozygous dystrophic (genotype, dydy) and heterozygous (genotype, Dydy) muscles of the house mouse. In adult heterozygous muscle and muscle of a Swiss albino mouse there was usually a reciprocal relationship between phosphorylase and lactate dehydrogenase, i.e., weak lactate dehydrogenase reactions in the majority of fibers of the vastus lateralis and a stronger phosphorylase reaction. Weak to strong lactate dehydrogenase reactions were identified in fibers of the rectus femoris. In heterozygous muscle of young mice 20 days of age, a reciprocal relationship between lactate dehydrogenase and phosphorylase was not observed. Reciprocity between phosphorylase and lactate dehydrogenase was not always evident in fibers of homozygous muscles, e.g., groups of homozygous fibers frequently exhibited both strong phosphorylase and lactate dehydrogenase reactions whereas other fibers reacted weakly for phosphorylase or not at all and strongly for lactate dehydrogenase. Small fibers reacted either weakly or strongly for dehydrogenases. One of the striking features which homozygous fibers usually showed was uniformly strong dehydrogenase reactions (e.g. lactate, glutamate, succinate) and diphosphopyridine nucleotide linked diaphorase reactions. Strong glucose-6-phosphate dehydrogenase reactions were frequently identified in homozygous dystrophic fibers whereas weak reactions or no reactions at all were seems in heterozygous fibers. Atrophic fibers of homozygous dystrophic muscles exhibited intense indoxyl acetate esterase and acid phosphatase reactions. Weak adenosine triphosphatase reactions were seen in fibers of both heterozygous and homozygous dystrophic muscle, and strong reactions in connective tissues and blood vessels. Interfibrillar connective tissue of homozygous muscle was abundant and exhibited intense acid phosphatase reactions, whereas connective tissues of heterozygous muscle were less concentrated and exhibited weak acid phosphatase reactions.