Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.6.99.1 (NADPH-diaphorase)
 3,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

It is generally thought that the oxidative modification of hemoproteins leads to their inactivation. In the current study, however, a transiently activated form of myoglobin was shown to be formed when the prosthetic heme group became covalently bound to the polypeptide during the reaction of myoglobin with low levels of HOOH. In the presence of an enzymatic metmyoglobin reducing system containing diaphorase and methylene blue with excess NADH, this HOOH-altered myoglobin catalyzed NADH oxidation and oxygen consumption; the overall stoichiometry indicated a two-electron reduction of oxygen to HOOH. This reaction was not catalyzed by iron released from heme, as desferrioxamine had no effect on the activity. Stoichiometric amounts of HOOH were sufficient to produce the activated oxidase state of myoglobin, whereas larger amounts of HOOH lead to heme destruction, iron release, and inactivation of the oxidase activity. The alteration of myoglobin to an enzyme that can form toxic oxygen metabolites may have pathological importance, especially in myocardial injury caused by ischemia and reperfusion, where myoglobin is present in large amounts and HOOH is formed. Furthermore, the oxidase form may be involved in the mechanism of destruction of the heme seen with oxidative treatment of myoglobin.
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PMID:Oxidative modification by low levels of HOOH can transform myoglobin to an oxidase. 187 Nov 23

D-Lactate in biological samples was converted into a strongly fluorescent substance in a one-vial reaction. It was first converted into the pyruvate hydrazone in the presence of D-lactate dehydrogenase, an NADH-reoxidation system using diaphorase, D,L-6,8-thioctamide and hydrazine. This hydrazone was then converted into 2-hydroxy-6,7-dimethoxy-3-methylquinoxaline by 1,2-diamino-4,5-dimethoxybenzene in 1 M hydrochloric acid, and the quinoxaline was extracted and measured fluorimetrically at 432 nm (excitation at 365 nm). The calibration curve for D-lactate was linear up to at least 100 nmol/ml of the assay mixture, with a determination limit of 2 nmol/ml. The quinoxaline was also analysed by high-performance liquid chromatography with fluorimetric detection. The calibration curve for D-lactate was linear from 500 fmol to 75 nmol in the reaction mixture. This method was 4000 times more sensitive than the fluorimetric method, and could determine D-lactate in blood plasma volumes of less than 1 microliter.
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PMID:Fluorimetric and high-performance liquid chromatographic determination of D-lactate in biological samples. 188 4

Endogenous activities of phospholipases A and C in Ureaplasma urealyticum were assayed in cellular fractions of exponential-phase cells. Enzymatic studies indicated that ATPase activity was localized in the plasma membrane fraction and NADH and NADPH dehydrogenase activities were localized in the cytosol fraction. Studies with purified ureaplasma membranes demonstrated that, of three serovars tested, endogenous phospholipase A1, A2, and C activities were localized in the plasma membrane. Very low levels of activity were observed in the cytosol fractions. Phospholipase A2 activity in the plasma membrane was 3- to 5-fold higher than the activity in the lysates and 60- to 300-fold higher than the activity of phospholipase A1. Phospholipase C was localized mainly in the plasma membrane, with 20% found in the cytosol fraction. The levels of activity were comparable among the three serovars. There was a significantly lower level of activity in cells from the stationary growth phase than in the exponential phase. Significant differences were observed in the phospholipase A activities among the U. urealyticum serovars 3, 4, and 8. Phospholipase A2 activity was twofold higher in serovar 8 membranes, and phospholipase A1 activity was twofold higher in serovar 3 membranes. These results demonstrate that endogenous activities of phospholipase A and C are localized primarily in the plasma membrane fraction of U. urealyticum. The specific activities in the membranes of the phospholipases varied among the three serovars. Phospholipase enzymes may function as virulence factors in U. urealyticum and may vary among the serovars.
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PMID:Localization of endogenous activity of phospholipases A and C in Ureaplasma urealyticum. 188 45

NAD(P)H: quinone oxidoreductase (NQO1) is believed to be protective against cancer and toxicity caused by exposure to quinones and their metabolic precursors. This enzyme catalyzes the two-electron reduction of compounds, compared with one-electron reduction mediated by NADPH: cytochrome-P450 oxidoreductase which produces toxic and mutagenic free radicals. Recently we cloned and sequenced the cDNA encoding human 2.3,7,8-tetrachlorodibenzo-p-dioxin (dioxin)-inducible cytosolic NQO1 [Jaiswal et al. (1988) J. Biol. Chem. 263, 13572-13578] and provided preliminary evidence that this enzyme may correspond to diaphorase 4, an enzymatic activity present in various tissues that catalyzes the reduction of a variety of quinones by both NADH and NADPH [Edwards et al. (1980) Biochem. J. 187, 429-436]. In the present report we characterize the catalytic properties of the protein encoded by the NQO1 cDNA. The enzyme was synthesized in monkey kidney COS-1 cells transfected with a pMT2-based expression plasmid containing the NQO1 cDNA. Western blot analysis of the transfected cells using an antibody against rat liver cytosolic NQO1 revealed a 31-kDa band that was not detected in nontransfected cells. This band corresponded to a polypeptide with the same electrophoretic mobility as the endogenous NQO1 protein detected in the human hepatoblastoma (Hep-G2) cells with the same antibody. The immunoreactive protein detected in human Hep-G2 cells was induced approximately fourfold by exposure of the cultures to dioxin, an increase commensurate with the increased in quinone oxidoreductase activity. These studies suggest that the protein encoded by NQO1 cDNA is indeed similar, if not identical, to the dioxin-inducible protein band detected in human Hep-G2 cells. Further characterization of the product of NQO1 cDNA, which was present at approximately 20-30-fold higher levels in transfected COS cells than the endogenous product in uninduced human Hep-G2 cells indicated that it had very high capacity (greater than 1000-fold over background) to catalyze the reduction of 2.6-dichloroindophenol and menadione. Besides these two commonly used substrates for quinone reductase, the expressed NQO1 protein also effectively metabolized 2,6-dimethylbenzoquinone, methylene blue, p-benzoquinone, 1,4-naphthoquinone, 2-methyl-1,4-benzoquinone, with the latter being the most potent electron acceptor at 50 microM concentration of the substrate.
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PMID:The human dioxin-inducible NAD(P)H: quinone oxidoreductase cDNA-encoded protein expressed in COS-1 cells is identical to diaphorase 4. 189 80

The interaction between lipoamide dehydrogenase (E3) and dihydrolipoyl transacetylase (E2p) from the pyruvate dehydrogenase complex was studied during the reconstitution of monomeric E3 apoenzymes from Azotobacter vinelandii and Pseudomonas fluorescens. The dimeric form of E3 is not only essential for catalysis but also for binding to the E2p core, because the apoenzymes as well as a monomeric holoenzyme from P. fluorescens, which can be stabilized as an intermediate at 0 degree C, do not bind to E2p. Lipoamide dehydrogenase from A. vinelandii contains a C-terminal extension of 15 amino acids with respect to glutathione reductase which is, in contrast to E3, presumably not part of a multienzyme complex. Furthermore, the last 10 amino acid residues of E3 are not visible in the electron density map of the crystal structure and are probably disordered. Therefore, the C-terminal tail of E3 might be an attractive candidate for a binding region. To probe this hypothesis, a set of deletions of this part was prepared by site-directed mutagenesis. Deletion of the last five amino acid residues did not result in significant changes. A further deletion of four amino acid residues resulted in a decrease of lipoamide activity to 5% of wild type, but the binding to E2p was unaffected. Therefore it is concluded that the C-terminus is not directly involved in binding to the E2p core. Deletion of the last 14 amino acids produced an enzyme with a high tendency to dissociate (Kd approximately 2.5 microM). This mutant binds only weakly to E2p. The diaphorase activity was still high. This indicates, together with the decreased Km for NADH, that the structure of the monomer is not appreciably changed by the mutation. Rather the orientation of the monomers with respect to each other is changed. It can be concluded that the binding region of E3 for E2p is constituted from structural parts of both monomers and binding occurs only when dimerization is complete.
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PMID:Interaction of lipoamide dehydrogenase with the dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii. 190 77

The distribution of acetylcholinesterase and NADH-diaphorase activities was studied histochemically in the rabbit medial geniculate body, yielding new data useful for the definition of the common structural pattern of this thalamic complex in mammals. Four chemoarchitectonic subdivisions could be detected in transversal, horizontal and sagittal sections that corresponded to the previously described ventral, dorsal and internal nuclei, and to a fourth subdivision, defined as the mediorostral nucleus of the medial geniculate complex in the rabbit. The topography and cellular typology of the mediorostral nucleus suggest its homology with the so-called magnocellular nucleus of other mammals, an identity that was previously assigned to the internal nucleus. The relative position of the rabbit internal and dorsal nuclei and comparative connectional data are combined to suggest their correspondence with the anterodorsal and posterodorsal subnuclei, respectively, of the cat and the monkey. Global functional interpretations of these nuclei as sites of visuoacoustic and somatoacoustic polymodal integration support the notion of a shell region of the medial geniculate, surrounding the principal cochleotopic ventral nucleus and interconnected to the cortical acoustic belt around the primary auditory area. Acetylcholinesterase and NADH-diaphorase chemoarchitectony may be useful for the detection of similar partitions in species where cytoarchitectonic differentiation of the medial geniculate is less clear.
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PMID:Acetylcholinesterase and NADH-diaphorase chemoarchitectonic subdivisions in the rabbit medial geniculate body. 193 Jul 48

The myenteric plexus of the domestic fowl (Gallus domesticus) small intestine was studied by means of silver staining, glyoxylic acid-induced fluorescence, the modified Koelle-Friedenwald method for the detection of acetylcholinesterase, NADH-diaphorase techniques and the unlabelled antibody method involving the use of an antiserum raised against GABA conjugated by glutaraldehyde to bovine serum albumin. The majority of the perikarya were in the ganglia, with an average density of 3370 +/- 942 nerve cells/cm2. Cholinesterase-positive and a few GABA-immunoreactive nerve cell bodies were seen in the myenteric ganglia, while fluorescent ganglion cells were not observed. In addition to AChE and GABA-positive nerve fibres, a rich fluorescent network of varicose and nonvaricose nerve fibres was detected, pointing to the presence of an extrinsic aminergic system in the domestic fowl myenteric plexus. Electron microscopic observations on nerve cells, axon profiles and varicosites with various vesicle populations were in good agreement with the histochemical findings.
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PMID:Histochemical characterization of myenteric plexus in domestic fowl small intestine. 207 64

Two forms of NADH-dependent oxidoreductase (diaphorase [EC.1.6.99.-]) are established in boar spermatozoa. The first form is typical for soluble proteins with a varying electrophoretic profile, while the other form for sedimental proteins with a specific, slowly-moving fraction, which is not common for the soluble form. The two enzyme forms have a close isoelectric point (pI5.5-6.0) and they can not be inhibited by dicumarol 10(-5) mol l-1 and FAD 10(-4) mol l-1. The molecular mass of the soluble form of the enzyme is 28, 37, 46 and 67 kD, while of the sedimental form it is 220, 250 and 260 kD, respectively.
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PMID:Electrophoresis of NADH-dependent oxidoreductase (diaphorase) in boar spermatozoa. 209 76

We describe an assay for light microscopic visualization of specific glycosyltransferases on tissue sections or on cells. The assay uses a sequence of enzyme reactions that yields two moles of NADH for each mole of the uridine-5'-diphosphate (UDP) released during transfer of a monosaccharide from a UDP sugar to an acceptor. When diaphorase and tetrazolium salts are present in the incubation mixture, the tetrazolium salts are reduced to colored diformazans, which precipitate at the sites of glycosyltransferase activity. The validity of the assay was established by applying the technique to spermatozoa and liver, in which some glycosyltransferases have previously been localized. When suspensions of mouse spermatozoa were assayed for galactosyltransferase (GalTase) activity, diformazan precipitates appeared on the plasma membranes overlying the anterior heads of the spermatozoa, in agreement with immunochemical localizations. In mouse liver slices assayed with bilirubin as acceptor for glucuronyltransferase (GluTase) activity, dense diformazan deposits appeared on the hepatocytes but not on endothelial cells, also in agreement with immunochemical data. In the absence of acceptor or UDP sugar donor, diformazan deposits were minimal and random in all tissues tested. The assay's versatility was tested by incubating tissues with different sugar donors and acceptors to localize other sites of transferase activity. In mouse frozen liver sections, GalTase activity occurred in both hepatocytes and endothelial cells; in sections of rat submaxillary glands, GalTase activity was detected in mast cells. In liver sections, GlcuTase activity with o-aminophenol as acceptor was located primarily on the endothelial cells. With the appropriate sugar donor and acceptor, this assay should detect any transferase, other than the glucosyltransferases, that utilizes UDP sugars.
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PMID:Light microscopic localization of glycosyltransferase activities in cells and tissues. 210 33

By crossed immunoelectrophoresis with antibodies against the NAD-linked hydrogenase the presence of three hydrogenase protein species was demonstrated in crude extracts of Alcaligenes eutrophus H16. Protein 1 (antigen 1) exhibited NAD-reducing activity and was shown to be identical with the native heterotetrameric enzyme. Protein 2 (antigen 2) was catalytically inactive in the antibody-precipitated form and corresponded to the beta subunit (56 kDa) of the holoenzyme. Protein 3 (antigen 3) was serologically distinct from antigen 2 and catalyzed NADH-oxidizing (diaphorase) activity, suggesting that it either consists of the alpha peptide or of the alpha and gamma subunits of the diaphorase dimer. Tandem immunoelectrophoresis revealed that antigen 2 was the predominant protein species in cells cultivated under nickel deficiency. Low concentrations of the diaphorase-active antigen 3 were also detected under these conditions. Extracts from mutants defective in the catalytic activity of NAD-reducing hydrogenase still contained the four polypeptides. This was shown by immunodiffusion and immunoblotting with antibodies raised against the individual subunits. However, as observed with nickel-deficient cells, no complete tetrameric protein could be identified, and the dominant subunit species (70-80%) was the beta peptide.
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PMID:Identification of distinct NAD-linked hydrogenase protein species in mutants and nickel-deficient wild-type cells of Alcaligenes eutrophus H16. 211 62


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