Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.6.99.3 (diaphorase)
5,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

NO synthase (NOS; EC 1.14.23) catalyzes the conversion of L-arginine into L-citrulline and a guanylyl cyclase-activating factor (GAF) that is chemically identical with nitric oxide or a nitric oxide-releasing compound (NO). Similar to the other isozymes of NOS that have been characterized to date, the soluble and Ca2+/calmodulin-regulated type I from rat cerebellum (homodimer of 160-kDa subunits) is dependent on NADPH for catalytic activity. The enzyme also possesses NADPH diaphorase activity in the presence of the electron acceptor nitroblue tetrazolium (NBT). We investigated the requirements of NOS and its content of the proposed additional cofactors tetrahydrobiopterin (H4biopterin) and flavins, further characterized the NADPH diaphorase activity, and quantified the NADPH binding site(s). Purified NOS type I Ca2+/calmodulin-independently bound the [32P]2',3'-dialdehyde analogue of NADPH (dNADPH), which, at near Km concentrations during 3-min incubations was utilized as a substrate and at higher concentrations or after prolonged incubations and cross-linking inhibited NOS activity. The NADPH diaphorase activity was Ca2+/calmodulin-independent, required higher NADPH concentrations than NOS activity, and was affected by dNADPH to a lesser degree. Divalent cations interfered with the diaphorase assay. Per dimer, native NOS contained about 1 mol each of H4biopterin, FAD, and FMN, classifying it as a biopteroflavoprotein, and incorporated 1 mol of dNADPH. No dihydrobiopterin (H2biopterin), biopterin, or riboflavin was detected. These findings suggest that NOS may share cofactors between two identical subunits via high-affinity binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Ca2+/calmodulin-dependent NO synthase type I: a biopteroflavoprotein with Ca2+/calmodulin-independent diaphorase and reductase activities. 137 27

O2- production by homogenates and isolated membranes of E. coli has been examined. Approximately one-fourth of the O2- generated by extracts in the presence of NAD (P) H is attributable to the membranes. The autoxidizable membrane component is a member of the respiratory chain, since O2- production is NADH-specific, amplified by cyanide, and absent from membranes lacking the respiratory NADH dehydrogenase. Other respiratory substrates (succinate, 1-phosphoglycerol, D-lactate, and L-lactate) supported O2-production at efficiencies between 3 and 30 O2- released per 10,000 electrons transferred, under conditions of substrate saturation. Membranes from quinoneless mutants quantitatively retain the ability to evolve O2-, indicating that the dehydrogenases are the sites of O2- production. Relative O2- production was greater at low substrate concentrations, probably reflecting the facilitation of unpairing of electrons that may occur when enzymes with multiple redox centers are only partially reduced. Respiration rate, cell volume, rates of membraneous and cytosolic O2- production, and SOD levels were used to calculate a steady-state concentration of O2- between 10(-10) and 10(-9) M in well-fed, aerobic, SOD-proficient cells.
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PMID:Superoxide production by respiring membranes of Escherichia coli. 164 4

One-electron reduction of diaziquone (AZQ) by purified rat liver NADPH cytochrome c reductase was associated with formation of AZQ semiquinone, superoxide anions, hydrogen peroxide, and hydroxyl radicals as indicated by ESR spin-trapping studies. Reactive oxygen formation correlated with AZQ-dependent production of single and double PM2 plasmid DNA strand breaks mediated by this system as detected by gel electrophoresis. Direct two-electron reduction of AZQ by purified rat liver NAD(P)H (quinone acceptor) oxidoreductase (QAO) was also associated with formation of AZQ semiquinone, superoxide anions, hydrogen peroxide, and hydroxyl radicals as detected by ESR spin trapping. Furthermore, PM2 plasmid DNA strand breaks were detected in the presence of this system. Plasmid DNA strand breakage was inhibited by dicumarol (49 +/- 5%), catalase (57 +/- 2.3%), SOD (42.2 +/- 3.6%) and ethanol (41.1 +/- 3.9%) showing QAO and reactive oxygen formation was involved in the PM2 plasmid DNA strand breaks observed. These results show that both one- and two-electron enzymatic reduction of AZQ give rise to formation of reactive oxygen species and DNA strand breaks. Autoxidation of the AZQ semiquinone and hydroquinone in the presence of molecular oxygen appears to be responsible for these processes. QAO appears to be involved in the metabolic activation of AZQ to free radical species. The cellular levels and distribution of this enzyme may play an important role in the response of tumor and normal cells to this antitumor agent.
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PMID:Free radical formation and DNA strand breakage during metabolism of diaziquone by NAD(P)H quinone-acceptor oxidoreductase (DT-diaphorase) and NADPH cytochrome c reductase. 166 2

Until recently, no data on genetic polymorphisms in the populations living on the northern side of the Pyrenees have been available, except for the Basques. Several investigations were done lately on rural communities in various geographic zones in the Pyrenees from the eastern to the western part. In this paper, the results for the following enzyme polymorphisms are reported: acid phosphatases, AK, ADA, PGM1 and PGM2, 6PGD, NADH diaphorase, SOD, MDH, TGP, G6PD, C5 esterase (E2 locus), serum cholinesterase (E1 locus). Significant variation in gene frequencies was observed over the distinct geographic zones for the main polymorphic system. Furthermore, some rare alleles were found: a new G6PD variant (Luz-Saint-Sauveur), the presence of ADA3 and ADA5 alleles in two groups of the Central Pyrenees, a Dia2 gene among Basques and in the Pays de Sault, a high rate of Ea1 allele in the Basque group. The values obtained for the degree of heterozygosity are in agreement with the relative isolation of the different groups studied and confirm the importance of sociocultural factors in the evolution of the genetic background of rural communities in Europe.
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PMID:Study of red blood cell and serum enzymes in five Pyrenean communities and in a Basque population sample. 644 18

Osteoclasts have been shown to destroy calcified tissue by complex developmental steps involving cell recruitment, cell attachment and deployment of multiple enzymes. They also appear to regulate resorption by several mechanisms. In particular, earlier investigations have indicated that oxygen radical metabolites may be produce by osteoclasts. These labile reactants could accelerate destruction of calcified tissue. In addition, recent studies have suggested that nitric oxide may have an inhibitory role in bone resorption. Previous studies of these radical substituents have predicted that interactions of nitric oxide and oxygen radicals could explain the conflicting roles of these radicals in the control of bone resorption. In view of the requirement of both of the enzymes, NADPH-oxidase and NO synthase (NOS), for NADPH(beta-nicotinamide adenine dinucleotide phosphate), one level of interaction could be related to competition for this necessary cofactor. To test this hypothesis, we have investigated the ability of the osteoclast to generate nitric oxide and oxygen radicals after stimulation by NADPH. Consistent with earlier diaphorase histochemistry, we have shown that resorbing osteoclasts produce NO. Addition of NADPH (10 microM) resulted in a transient burst of NO production (measured by porphyrin coated microsensor) with an amplitude of 152 +/- 43 nM and a duration of 4 seconds. Repetitive stimulation resulted in a decremental response with a partial recovery after 30 minutes. Addition of L-NAME (N omega-nitro-L-arginine methyl ester, 100 microM) to the cells resulted in at least 50% inhibition of the amplitude of NO peak and produced an extended peak duration. To compare the effect of the added NADPH on superoxide production by osteoclast NADPH-oxidase, osteoclast oxygen radicals were detected by EPR(electron paramagnetic resonance) spectrometer with the spin-trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The production of a spin adduct with a quadruplet signal was inhibited by SOD (superoxide dismutase). We were not able to demonstrate an increase in superoxide production after addition of L-NAME, another possible interaction of NOS and NADPH-oxidase. These results demonstrate that although osteoclasts produce both NO and superoxide, NOS competition for NADPH is not a major site of interaction with NADPH-oxidase under these conditions. Additionally, these initial findings set the stage for the further investigation of interactions of osteoclast radicals in modulating bone resorption.
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PMID:Osteoclast radical interactions: NADPH causes pulsatile release of NO and stimulates superoxide production. 758 66

Manganese superoxide dismutase (Mn SOD), mitochondrial enzyme, defends against the toxic effects of superoxide radical (O2.-) in pathological processes by catalyzing the conversion of O2.- to hydrogen peroxide (H2O2). The activity of another enzyme, NADH diaphorase, forms the basis for a histochemical method used commonly to demonstrate nerve cell bodies in the enteric plexuses. We found identical patterns of localization of Mn SOD immunoreactivity and NADH diaphorase activity in brain, esophagus, stomach, colon, liver, and kidney. NADH diaphorase enzymatic activity co-migrated with complexes of Mn SOD on a non-denaturing gel. This suggests that the NADH diaphorase may in some way be related to Mn SOD.
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PMID:Co-localization of manganese superoxide dismutase and NADH diaphorase. 762 45

NADPH diaphorase activity was found in membrane of DMSO-induced differentiated human promyelocytic leukemia HL-60 cells. This membrane-bound diaphorase activity increased dramatically during differentiation of HL-60 cells. A dye reductase was extracted from membrane of DMSO-induced differentiated HL-60 cells with n-octyl glucoside and sodium cholate in the presence of several protease inhibitors such as PMSF, DIFP, TLCK, antipain, chymostatin, leupeptin, pepstatin A and trypsin inhibitor. The NADPH diaphorase was highly purified by two-stage sequential column chromatographies. The purified enzyme, showing both SOD-insensitive cytochrome c and NBT reductase activities, migrated with an apparent molecular mass of 77 kDa on SDS-PAGE. When the purification of this diaphorase was carried out in the presence of only three protease inhibitors, PMSF, DIFP and TLCK, a partially proteolyzed form of the diaphorase with a molecular mass of 68 kDa was prepared. The proteolyzed diaphorase exhibited only an NADPH-dependent cytochrome c reductase. The NADPH diaphorase gave a positive cross-reaction to polyclonal antibodies raised against microsomal NADPH-cytochrome P450 reductase from rabbit liver.
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PMID:Purification of an NADPH-dependent diaphorase from membrane of DMSO-induced differentiated human promyelocytic leukemia HL-60 cells. 769 24

Superoxide dismutase-like activity (SOD-like), isoenzyme lactate dehydrogenase-C4 (LDH-C4) and NADH-diaphorase activities in spermatozoa have been investigated from 58 normozoospermic and 27 oligozoospermic men. Significantly higher SOD-like, LDH-C4 and diaphorase activities (P < 0.01, P < 0.005 and P < 0.0001, respectively) were detected in spermatozoa from oligozoospermic men, compared to the activities found in normozoospermic samples. SOD-like activity (mean +/- SE) in oligozoospermic samples amounted to 8.3 +/- 1.6 U 10(-8) spermatozoa, while in spermatozoa in normozoospermic men with a sperm concentration above 20 million of spermatozoa per ml amounted to 4.2 +/- 0.5 U 10(-8). There was a close correlation between the SOD-like activity and biochemical indicators of the presence of residual cytoplasm i.e. isoenzyme LDH-C4 and NADH-diaphorase (r = 0.53 and r = 0.66 in normozoospermic and r = 0.63 and r = 0.54 in oligozoospermic men, respectively). A positive relationship between SOD-like activity and experimentally-induced lipid peroxidation was detected in 54 infertile men (r = 0.30; P < 0.05). These findings suggest that a higher level of superoxide dismutase-like activity may reflect a defect in the development or maturation of spermatozoa and, thereby, a decreased fertility potential. Hence, determination of SOD-like activity may give information on the state of maturity of human spermatozoa, while its role in the antioxidative protection remains to be determined.
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PMID:Relationship of sperm superoxide dismutase-like activity with other sperm-specific enzymes and experimentally induced lipid peroxidation in infertile men. 884 16

The occurrence of NADH --> NAD transhydrogenation and lipoamide dehydrogenase activities was demonstrated for cysticercoids of the intestinal cestode, Hymenolepis diminuta. In addition, both activities were catalyzed by the mitochondria of 6-, 10-, and 14-day H. diminuta and by the mitochondria from immature, mature, and pregravid/gravid regions of the adult cestode. A developmentally related increase in NADH --> NAD activity was suggested and the levels of both activities in the immature region of the helminth were consistent with it being a region of high metabolic activity. Adult H. diminuta mitochondrial lipoamide dehydrogenase was purified to homogeneity. The native enzyme was a homodimer with a monomeric and dimeric molecular mass of 47 and 93 kDa, respectively. Spectral analyses revealed that the enzyme contained flavin. More importantly, the purified enzyme catalyzed appreciable NADH --> NAD transhydrogenation activity, a premier finding for the phylum Platyhelminthes. The ratio of NADH --> NAD transhydrogenation to lipoamide reduction was 1:5. Both activities were inhibited by Cu2+ and Cd2+ with the NADH --> NAD activity being more resistant to inhibition. Interestingly, aside from NADH diaphorase activity, the cestode enzyme displayed NADH-ferricyanide reductase and, to a lesser degree, NADPH --> NAD transhydrogenation activities. The partial amino acid sequence of H. diminuta lipoamide dehydrogenase indicated that this enzyme was most similar to the corresponding enzymes of other parasitic helminths. Moreover, the phenylalanine for leucine substitution found in the redox-active disulfide site of the lipoamide dehydrogenases of some anaerobic systems was noted for the H. diminuta enzyme.
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PMID:Hymenolepis diminuta: mitochondrial NADH --> NAD transhydrogenation and the lipoamide dehydrogenase system. 903 Jun 66

The quaternary behaviour of DT diaphorase in solution has been investigated by hydrodynamics under a range of conditions. At neutral pH DT diaphorase is shown to exist as a tightly-associated homodimer in a dimer-tetramer equilibrium. Concentrations of the chaotropic agent potassium thiocyanate (KSCN) of greater than 200 mM result in irreversible loss of the FAD cofactor and denaturation of the homodimer though this agent appears to be ineffective in disrupting intermolecular association. These data conform to a model in which under extreme dissociation conditions, the folded dimer is in equilibrium with the unfolded monomer and are consistent with evidence from the X-ray structure and proposed catalytic mechanism where both monomers are catalytically interdependent.
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PMID:DT diaphorase exists as a dimer-tetramer equilibrium in solution. 918 64


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