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:1.6.99.1 (NADPH-diaphorase)
3,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Techniques for the ultrastructural demonstration of dehydrogenases in cerebral cortex are described. The best fixation for good fine structural preservation and retention of LDH and NADH-diphorase was obtained by perfusion with a misture of formaldehyde and glutaraldehyde and for SDH by perfusion with formaldehyde. Comparison of incubation conditions showed that consistent results were obtained using enzyme markers NBT and DS-NBT for LDH and NADH-diaphorase: DS-NBT was more satisfactory than NBT and BSPT for SDH. Penetration of incubation media was improved by Triton X-100: DMSO and ultrasonic treatment were less effective. The techniques enabled the first electron cytochemical demonstration of dehydrogenases in different elements of prefixed cerebral cortex. Ultrastructural sites of enzyme activities were localized within cristae and inter-membrane spaces of mitochondria in nerve cell cytoplasm and its processes, oligodendrocytes and astrocytes. Authenticity of the ultrastructural sites was confirmed by four different control experiments.
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PMID:Ultrastructural demonstration of dehydrogenases in rat cerebral cortex. 47 91

An NADPH-dependent membrane-bound flavoprotein dehydrogenase, assayed as a catalyst of electron transfer from NADPH to cytochrome c, was extracted from membranes of rabbit peritoneal neutrophils with Triton X-100 and sodium deoxycholate in the presence of diisopropylfluorophosphate as antiprotease, and purified to electrophoretic homogeneity. The purified enzyme in detergent was able to enhance the rate of formation of the superoxide anion O2- in a cell-free system, consisting of membrane and cytosolic fractions from resting neutrophils complemented with arachidonic acid, guanosine 5'-[gamma- thio]triphosphate and Mg2+. This suggested that the NADPH dehydrogenase was a component of the rabbit neutrophil oxidase complex. The purification factor of the enzyme with respect to the membrane fraction was close to 1000 and the recovery of activity was 33%. FMN and FAD were associated with the enzyme in a molar ratio close to 1. On SDS/PAGE, the enzyme migrated with a molecular mass of 77 kDa. A similar mass was determined by filtration on a molecular sieve. The isoelectric point of this enzyme was 4.7 +/- 0.1. Its activity was maximal between pH 7.5 and pH 8.5, and depended on the ionic strength of the medium, with a maximum at an ionic strength of 0.5. Reduction of cytochrome c by NADPH obeyed Michaelis-Menten kinetics with a KM value of 15 microM for cytochrome c. When NADPH was the variable substrate, a KM value of 1.9 microM for NADPH was found, but a significant deviation from Michaelis-Menten kinetics was observed at high concentrations of NADPH. Mersalyl strongly inhibited the reductase activity when added to the enzyme prior to NADPH; preincubation of the enzyme with NADPH considerably reduced the inhibitory efficiency of mersalyl. A partially proteolyzed water-soluble, active, form of enzyme with a molecular mass of 67 kDa was prepared. The proteolyzed enzyme exhibited the same specificity, and kinetic behavior with respect to NADPH, and the same dependency on the ionic strength, as the native enzyme.
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PMID:NADPH-cytochrome c reductase from rabbit peritoneal neutrophils. Purification, properties and function in the respiratory burst. 184 86

Superoxide (.O2-) production by the NADPH oxidase of a membrane fraction derived from rabbit peritoneal neutrophils activated by 4 beta-phorbol 12-myristate 13-acetate (PMA) was studied at 25 degrees C under different conditions, and measured by the superoxide dismutase inhibitable reduction of cytochrome c. Whereas PMA-activated rabbit neutrophils incubated in a glucose-supplemented medium exhibited a substantial rate of production of .O2-, the membranes prepared by sonication of the activated neutrophils were virtually unable to generate .O2- in the presence of NADPH. Instead, they exhibited an NADPH-dependent diaphorase activity, measured by the superoxide-dismutase-insensitive reduction of cytochrome c. Upon addition of arachidonic acid, which is known to elicit oxidase activation, the NADPH diaphorase activity of the rabbit neutrophil membranes vanished and was stoichiometrically replaced by an NADPH oxidase activity. The emerging oxidase activity was fully sensitive to iodonium biphenyl, a potent inhibitor of the respiratory burst, whereas the diaphorase activity was not affected. Addition of 0.1% Triton X-100 or an excess of arachidonic acid, acting as detergent, resulted in the reappearance of the diaphorase activity at the expense of the oxidase activity. These results indicate that the diaphorase-oxidase transition is reversible. When the rabbit neutrophil membranes were supplemented with rabbit neutrophil cytosol, guanosine 5'-[gamma-thio]triphosphate and Mg2+, in addition to arachidonic acid, not only the NADPH diaphorase activity disappeared, but the emerging NADPH oxidase activity was markedly enhanced (about 10 times compared to that of membranes treated with arachidonic acid alone). The diaphorase-oxidase transition was accompanied by a 10-fold increase in the Km for NADPH, suggesting a change of conformation propagated to the NADPH-binding site during the transition. The treatment of PMA-activated rabbit neutrophils with cross-linking reagents, like glutaraldehyde or 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide, prevented the loss of the PMA-elicited oxidase activity upon disruption of the cells by sonication, suggesting that the interactions between the components of the oxidase complex are stabilized by cross-linking.
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PMID:Respiratory burst of rabbit peritoneal neutrophils. Transition from an NADPH diaphorase activity to an .O2(-)-generating oxidase activity. 217 79

NADH-cytochrome b5 reductase is the predominant NADH-diaphorase found in the human neutrophil (Blood 62:152, 1983). Although this reductase segregates with the light membranes of nitrogen-cavitated neutrophils separated on Percoll gradients (which include the plasma membrane markers alkaline phosphatase and NADPH-oxidase), it is approximately 95% excluded from plasma membrane-enriched phagocytic vacuoles. The reductase constitutes approximately 5% of the light membrane fraction FAD-flavoprotein (14.8 +/- 5.5 pmol/mg protein) and was found in equimolar concentration with a high potential b cytochrome also present in this light membrane fraction and tentatively identified as cytochrome b5. Isolation of the reductase from human neutrophils was accomplished by Triton X-114 solubilization of the light Percoll gradient membranes, followed by temperature-dependent phase separation and then affinity chromatography on AMP-Sepharose. The active preparation contained 1.3 mol FAD/mol protein, migrated on sodium dodecyl sulfate-polyacrylamide gels as a single band corresponding to an apparent mol wt of 45,000 daltons, exhibited a pl of 5.7 on chromatofocusing and was obtained in greater than 70% yield, with an overall purification of almost 900-fold. The purified enzyme was characterized by a high specificity for NADH as electron donor (Km = 6.4 mumol/L v Km greater than 1.6 mmol/L for NADPH) and exhibited a maximal turnover of ca. 30,000 min-1 at 22 degrees C with either ferricyanide or cytochrome b5 (Km = 10 nmol/L) as electron acceptor. Although the physical characterization and biochemical properties described here demonstrate that this neutrophil NADH b5 reductase is similar to the corresponding liver and erythrocyte enzymes, its unique function in the neutrophil has yet to be determined.
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PMID:Purification and characterization of the human neutrophil NADH-cytochrome b5 reductase. 299 39

Investigations were carried out into the activity and localization of NADH-dependant diaphorase in boar spermatozoa. Semen samples were collected from healthy boars, used in A.I. centers. The enzyme was extracted with distilled water and Triton X-100. Two forms of diaphorase were found-water-soluble and Triton X-100 soluble, showing low activity-0.36 U/ml and 0.26 U/ml. The enzyme was localized in the mitochondria, manifesting different intensities of reaction between sperm cells in the same ejaculate. It was found, that a part of the mitochondria and outer doublets showed positive reaction. It is suggested that the enzyme regulates the ratio between reduced and oxidized forms of NADH, takes part in the energy balance and possibly in the mechanism of sperm motility.
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PMID:Activity and localization of NADH-dependant oxidoreductase (diaphorase) in boar spermatozoa. 366 51

Monospecific rabbit antibodies against the ferredoxin-NADP+ reductase binding protein of spinach thylakoids were obtained and characterized. The immunoglobulin G (IgG) fraction gave single precipitation arcs with the purified antigen or with Triton X-100 extracts of thylakoids or the reductase binding protein complex. Antibodies against the flavoprotein behave similarly. Both antibodies agglutinated thylakoids and precipitated the diaphorase activity of a Triton X-100 extract of these membranes. Isolated Fab fragments of the IgG anti-binding protein inhibited NADP+ photoreduction in a time- and Fab concentration-dependent manner. The presence of ferredoxin diminished the rate of inhibition. In the light, the inactivation rate was higher than in dark and this effect was abolished in the presence of uncouplers. These results suggest that the binding protein is protruding from the thylakoids and could be sensing the proton gradient.
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PMID:Immunological studies of the binding protein for chloroplast ferredoxin-NADP+ reductase. 381 68

Bulk membrane fragments were prepared from cells of Bacillus cereus ATCC 4342 harvested at different stages of growth and sporulation and examined for enzymes involved in electron transport functions. The presence of succinate: DCPIP oxidoreductase (EC 1.3.99.1), succinate: cytochrome c oxidoreductase (EC 1.3.2.1), NADH:DCPIP oxidoreductase (EC 1.6.99.1), NADH:cytochrome c oxidoreductase (EC 1.6.2.1), succinate oxidase [succinate: (O(2)) oxidoreductase, EC 1.3.3.1], and NADH oxidase [NADH:(O(2)) oxidoreductase, EC 1.6.3.1] were demonstrated in membrane fragments from vegetative cells, early and late stationary-phase cells, and in cells undergoing sporulation. During the transition from a vegetative cell to a spore, there was a significant increase in the levels of enzymes associated with energy production via the electron transport system. Cytochromes of the a, b, and c type were detected in all membrane preparations; however, there was a marked increase in the level of cytochromes by the end of vegetative growth which remained throughout sporulation; there were no qualitative changes in the cytochromes throughout growth and sporulation. Sporulation was inhibited by cyanide, stressing the significance of the electron transport system. Enzyme activities were partially masked in washed membrane fragments; however, unmasking (stimulation) was achieved by sodium deoxycholate, sodium dodecyl sulfate, or Triton X-100. The degree of enzyme masking was less in vegetative cell membrane fragments than in membranes prepared from stationary-phase or sporulating cells. Results indicate the development of a membrane-bound electron transport system in B. cereus by the end of growth and prior to sporulation, which results in an increased masking of a number of enzymes associated with the terminal respiratory system of the cell.
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PMID:Development of a membrane-bound resiratory system prior to and during sporulation in Bacillus cereus and its relationship to membrane structure. 433 50

A membrane-associated b-type cytochrome (a proposed component in the neutrophil microbicidal superoxide generating system) has been partially purified from nonactivated beef granulocytes to a specific heme content of 20 nmol of heme/mg of protein, a value about 10-fold higher than those previously reported. The hemoprotein was solubilized at low temperature (4 degrees C) from mixed granule (30,000 X g) cell fractions using Triton X-114 detergent. Warming the extract to 25 degrees C allowed separation into detergent and aqueous phases; cytochrome b558 partitioned exclusively into the detergent phase, allowing separation from other visible-absorbing species (e.g. myeloperoxidase) and indicated an intrinsic membrane localization (Bordier, C. (1981) J. Biol. Chem. 256, 1604-1607). The partitioned cytochrome was chromatographed on hydroxylapatite and a hydrophobic affinity matrix, allowing a 185-fold (heme content) purification from the granule extract. The cytochrome preparation revealed three equal-staining protein bands by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis; apparent molecular weights were 14,000, 12,000, and 11,000. The question of heterogeneity of the preparation versus subunit structure is not resolved at present. The hemoprotein binds carbon monoxide, consistent with a proposed role as a terminal oxidase, and has an unusually negative oxidation-reduction potential (-225 mV) similar to that observed in granulocyte membranes. The preparation is devoid of NAD(P)H-diaphorase and cytochrome c reductase activities.
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PMID:Cytochrome b558 from (bovine) granulocytes. Partial purification from Triton X-114 extracts and properties of the isolated cytochrome. 643 85

The ferredoxin-NADP+ oxidoreductase of spinach chloroplasts was purified from a Triton X-100 thylakoid extract closely associated with an intrinsic polypeptide of 17.5 kDa. The 17.5-kDa polypeptide-reductase complex differs from soluble ferredoxin-NADP+ reductase in (a) its elution profile in an Affi-Gel blue column; (b) its behavior in isoelectric focusing electrophoresis; and (c) giving different immunoelectrophoretic arcs. The diaphorase activity of the purified complex showed the same pH profile of thylakoid-bound reductase. The curve changed to a form similar to that of soluble reductase after dissociation of the complex. Dissociation allowed separation of the components and was reversible. It is suggested that the 17.5-kDa intrinsic polypeptide is the reductase-binding protein and that it may play an important role in the physiological regulation of the reductase and of photosynthetic electron transport.
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PMID:Evidence for the existence of a thylakoid intrinsic protein that binds ferredoxin-NADP+ oxidoreductase. 673 31

The activity of reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) was examined histochemically in the rat cerebellar cortex at the light and electron microscopical level. Different staining patterns were observed depending on the technique used. Electron dense reaction product was seen on distinct membrane portions of the endoplasmic reticulum including the nuclear envelope in most of the neurons and in endothelial cells. Electron microscopically no activity staining was seen in glial cells, including Bergmann cells. The addition of the detergent Triton X-100, usually applied in the light microscopical diaphorase histochemistry, led to a striking diminution in membrane staining. In such preparations inspected electron microscopically formazan formed small granules which were evenly distributed over the cytoplasm.
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PMID:Localization of NADPH-diaphorase/nitric oxide synthase activity in the rat cerebellar cortex: a light and electron microscopical study. 753 9


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