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:1.8.1.4 (diaphorase)
2,754 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

The P, H, and T proteins of the glycine cleavage system have been purified separately from pea leaf mitochondria and demonstrate molecular weights of 98,000, 15,500, and 45,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of P protein by gel filtration was 210,000, indicating that this enzyme has a native homodimer conformation. Reconstitution assays containing purified P, H, and T proteins and yeast lipoamide dehydrogenase catalyze the oxidation of glycine and demonstrate a strict dependence on pyridoxal phosphate, tetrahydrofolate, NAD+, and dithiothreitol. The released CO2, methylamine-H protein intermediate, and methylenetetrahydrofolate are produced in stoichiometric amounts from glycine during the cleavage reaction. H protein acts as co-substrate with glycine during the decarboxylation reaction, demonstrating an apparent Km value of 2.2 microM. P and H protein alone jointly catalyze the glycine carboxyl-14 CO2 exchange reaction in the presence of pyridoxal phosphate and dithiothreitol. L protein of the glycine cleavage system was immunopurified using monoclonal antibodies. Antigenic and molecular weight similarities of the L protein with the lipoamide dehydrogenase component of the pyruvate dehydrogenase complex were shown suggesting the possibility of common isomers of lipoamide dehydrogenase for the two enzyme complexes.
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PMID:Glycine decarboxylase multienzyme complex. Purification and partial characterization from pea leaf mitochondria. 308 Apr 33

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

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

Inactivation of lipoamide dehydrogenase (LipDH) by the Cu(II)/H2O2 Fenton system (SF-Cu(II): (5.0 microM Cu(II), 3.0 mM H2O2) was enhanced by catecholamines (CAs), namely, epinephrine, levoDOPA (DOPA), DOPAMINE, 6-hydroxyDOPAMINE (OH-DOPAMINE) and related compounds (DOPAC, CATECHOL, etc.). After 5 min incubation with the Cu(II)/H2O2/CA system (0.4 mM CA), the enzyme activity decayed as indicated by the following percentage values (mean +/- S.D.; in parenthesis, number of determinations): SF-Cu(II) alone, 43 +/- 10 (18); SF-Cu(II) + epinephrine, 80 +/- 9 (5); SF-Cu(II) + DOPA, 78 +/- 2 (4); SF + Cu(II) + DOPAMINE, 88 +/- 7 (5); SF-Cu(II) + OH-DOPAMINE 87 +/- 6 (7); SF-Cu(II) +/- DOPAC, 88 +/- 3 (6); SF-Cu(II) + catechol, 85 +/- 6 (5). In all cases P < 0.05, with respect to the SF-Cu(II) control sample. CAs effect was concentration-dependent and at the 0-100 microM concentration range, it varied with the CA structure. Above the 100 microM concentration, CAs were equally effective and produced 90-100% enzyme, inactivation (Figure 2). In the absence of oxy-radical generation, the enzyme specific activity (mean +/- S.D.) was 149 +/- 10 (24) mumol NADH/min/mg protein. Assay of HO. production by the Cu(II)/H2O2/CA system in the presence of deoxyribose (TBA assay) yielded values much greater than those obtained omitting CA. Hydroxyl radical production depended on the presence of Cu(II) and H2O2 and significant H. values were obtained with OH-DOPAMINE, DOPAC, epinephrine, DOPAMINE, DOPA and catecol supplemented systems (Table 2). LipDH (1.0 microM) inhibited 50-80% deoxyribose oxidation, the inhibition depending on the CA structure (Table 2). Native catalase (20 micrograms/ml) and bovine serum albumin (40 micrograms/ml) effectively prevented LipDH inactivation by the Cu(II)/H2O2/CA system; denaturated catalase, SOD, 0.3 M mannitol, 6.0 mM ethanol and 0.2 M benzoate were less effective or did not protect LipDH (Table 3). Incubation of CAs with the Cu(II)/H2O2 system produced a time and Cu(II)-dependent destruction of CAs, the corresponding o-quinone, production as illustrated with epinephrine (figures 6 and 7), as illustrated with epinephrine and DOPAMINE (Table 4). These results support LipDH inactivation by (a) reduction of Cu(II) to Cu(I) by CAs followed by Cu-catalyzed production of HO. from H2O2; (b) CA oxidation followed by the corresponding o-quinone interaction with LipDH. CAPTOPRIL, N-acetylcysteine, mercaptopropionylglycine and penicillamine prevented to various degree LipDH inactivation by the Cu(II)/H2O2/CA systems (Table 1). The former was the most effective and 0.4 mM CAPTOPRIL prevented about 95-100% the effect of Cu(II)/H2O2/CA systems supplemented with epinephrine, DOPAMINE and OH-DOPAMINE (Figures 3 and Table 1). LipDH increased and CAPTOPRIL inhibited epinephrine oxidation by Cu(II)/H2O2 (Figures 4 and 5). Since un-physiological concentrations of CAs and Cu(II) may be released in the myocardium after ischemia-reperfusion, the summarized observations may contribute to explain myocardial damage in that condition.
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PMID:[Inactivation of the myocardial lipoamide dehydrogenase by catecholamines. Prevention by captopril and other thiol compounds]. 872 69

The enzymatic features and molecular species of the inhibitory action of menadione on lipid peroxidation in rat liver microsomes were examined. In an ascorbate-supported system or a NADH-supported reconstituted system containing NADH-cytochrome b5 reductase and cytochrome b5, menadione was not an inhibitor of lipid peroxidation at pH 7.5, while some antioxidant ability was observed at lower pH ranges. Lipid peroxidation in the presence of menadione in the NADH-supported reconstituted system at pH 7.5 was markedly inhibited by adding lipoamide dehydrogenase. NAD(P)H-supported lipid peroxidation in microsomes with increased DT-diaphorase activity from 3-methylcholanthrene-treated rats was highly susceptible to menadione. These inhibitions were abolished by dicoumarol, an inhibitor of DT-diaphorase. Cumene hydroperoxide-dependent lipid peroxidation in microsomes, with desferal and NADP+ to prevent nonheme iron-dependent reactions and oxygen radical generation, was inhibited by menadione in the presence of NADPH, and the inhibition was also more effective in the microsomes with increased DT-diaphorase activity. Menadiol reacted with 1,1-diphenyl-2-picrylhydrazyl (DPPH) in ethanol at a molar ratio of DPPH/menadiol at 1.9. In an iron-supported reconstituted enzymatic or a nonenzymatic system at pH 7.5, menadiol showed an antioxidant effect at an early stage, followed by a prooxidant effect, which was prevented by SOD, probably by protecting menadiol autooxidation. These results show that menadione exerts an antioxidant effect through participation of microsomal DT-diaphorase by generating menadiol with a radical scavenging ability, while menadiol also has a prooxidant property.
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PMID:Enzymatic and molecular aspects of the antioxidant effect of menadione in hepatic microsomes. 883 52

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

Two distinct dihydrolipoamide dehydrogenases (E3s, EC 1.8.1.4) have been detected in pea (Pisum sativum L. cv. Little Marvel) leaf extracts and purified to at or near homogeneity. The major enzyme, a homodimer with an apparent subunit M(r) value 56,000 (80-90% of overall activity), corresponded to the mitochondrial isoform studied previously, as confirmed by electrospray mass spectrometry and N-terminal sequence analysis. The minor activity (10-20%), which also behaved as a homodimer, copurified with chloroplasts, and displayed a lower subunit M(r) value of 52,000 which was close to the M(r) value of 52,614 +/- 9.89 Da determined by electrospray mass spectrometry. The plastidic enzyme was also present at low levels in root extracts where it represented only 1-2% of total E3 activity. The specific activity of the chloroplast enzyme was three- to fourfold lower than its mitochondrial counterpart. In addition, it displayed a markedly higher affinity for NAD+ and was more sensitive to product inhibition by NADH. It exhibited no activity with NADP+ as cofactor nor was it inhibited by the presence of high concentrations of NADP+ or NADPH. Antibodies to the mitochondrial enzyme displayed little or no cross-reactivity with its plastidic counterpart and available amino acid sequence data were also suggestive of only limited sequence similarity between the two enzymes. In view of the dual location of the pyruvate dehydrogenase multienzyme complex (PDC) in plant mitochondria and chloroplasts, it is likely that the distinct chloroplastic E3 is an integral component of plastidic PDC, thus representing the first component of this complex to be isolated and characterised to date.
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PMID:Identification and purification of a distinct dihydrolipoamide dehydrogenase from pea chloroplasts. 890 6

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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