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)

Ferredoxin-NADP+ reductase from Anabaena sp. PCC 7119 is chemically modified by pyridoxal 5'-phosphate. The incorporation of 2 +/- 0.3 mol pyridoxal 5'-phosphate/mol ferredoxin-NADP+ reductase inhibited NADPH-cytochrome c reductase activity by up to 95% while 55% of diaphorase activity still remained. Considerable protection against inactivation was afforded by ferredoxin. Chymotryptic cleavage of the modified enzyme was performed, the peptides were separated by high performance liquid chromatography, and the peptides containing pyridoxamine 5'-phosphate were identified by their fluorescence and by their absorbance at 325 nm. Three major labelled peptides were found. Their sequences were comprised of residues 46-54, 231-235 and 289-295. Lys-53 and -294 were the residues which presented the highest degree of modification and seem to be involved in the ferredoxin binding site of ferredoxin-NADP+ reductase from Anabaena sp. PCC 7119.
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PMID:Lysine residues on ferredoxin-NADP+ reductase from Anabaena sp. PCC 7119 involved in substrate binding. 154 17

Eleven independent monoclonal antibodies, all IgG's, have been raised against the ferredoxin:NADP+ oxidoreductase of spinach leaves. All 11 monoclonal antibodies were able to produce substantial inhibition of the NADPH to 2,6-dichlorophenol indophenol (DCPIP) diaphorase activity of the enzyme, but none of the antibodies produced any significant inhibition of electron flow from NADPH to ferredoxin catalyzed by the enzyme. Spectral perturbation assays were used to demonstrate that antibody interaction with NADP+ reductase did not interfere significantly with the binding of either ferredoxin or NADP+ to the enzyme. Ultrafiltration binding assays were used to confirm that the monoclonal antibodies did not interfere with complex formation between ferredoxin and the enzyme. These results have been interpreted in terms of the likely presence of one or more highly antigenic epitopes at the site where the nonphysiological electron acceptor, DCPIP, binds to the enzyme. Furthermore, the results suggest that the site where DCPIP is reduced differs from both of the two separate sites at which the two physiological substrates, ferredoxin and NADP+/NADPH, are bound.
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PMID:Monoclonal antibody studies of ferredoxin:NADP+ oxidoreductase. 165 83

This study identifies the neuronal types of the rhesus monkey lateral entorhinal cortex (LEC) and discusses the importance of these data in the context of the connectional patterns of the LEC and the possible role of these cells in neurodegenerative diseases. These neuronal types were characterized with the aid of Golgi impregnation techniques. These characterizations were based upon their spine densities, dendritic arrays, and, where possible, axonal arborizations. The cells could be segregated into only spinous and sparsely spinous types. The most numerous spinous types were pyramidal neurons. Other spinous types included multipolar, vertical bipolar and bitufted, and vertical tripolar neurons. The sparsely spinous neuronal types consisted of multipolar, horizontal bipolar and bitufted, and neurogliaform cells. These cells were further classified with the aid of histochemical stains and immunocytochemical markers. Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry stained multipolar, bipolar, and bitufted neurons. Stain for cytochrome oxidase (CO) was found in pyramidal and nonpyramidal cell types. Immunocytochemical techniques revealed several nonpyramidal neurons that contain somatostatin (Som) or substance P (SP). This study complements previous analyses of the neuronal components described in the LEC and adds further information about the distribution of selected neurochemicals within this cortex.
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PMID:Neurons of the lateral entorhinal cortex of the rhesus monkey: a Golgi, histochemical, and immunocytochemical characterization. 169 46

The activity of ferredoxin: NADP+ reductase (FNR) was found to decline to approximately 20% maximal levels with little or no loss in enzyme levels when cultures of the cyanobacterium Anabaena variabilis were maintained in the stationary phase of growth. Re-activation of enzyme activity occurred when cells were diluted into either fresh or re-utilized media and illuminated. This reversible de-activation/re-activation process was found, in vivo, to be dependent on the intensity of light illuminating the cells. The de-activated form of FNR was purified to homogeneity and exhibited the same molecular mass, isoelectric-focusing pattern and N-terminal amino acid sequence as the native form. Both de-activated and native FNR preparations each exhibited three reactive thiol groups on denaturation in urea; however, the rate of reaction with Ellman's reagent was much faster with the de-activated form than with the native form. Both preparations contain a single disulphide bond. Upon reduction of the disulphide bond in either form of the enzyme, the five reactive thiol groups exhibited identical reactivities in the presence of urea. Steady-state kinetic analysis of the de-activated form showed a marked increase in Km values for NADPH in diaphorase assays and an increase in Km for ferredoxin in the ferredoxin-mediated reduction of cytochrome c. No significant difference in kcat. was observed in comparison of the de-activated with the native form in any of the above assays; however, the de-activated form did exhibit a lower kcat. value in the transhydrogenase assay. The de-activated form of FNR bound ferredoxin with a 16-fold lower affinity than the native enzyme. These data suggest that the de-activation of FNR in vivo in response to low light intensity involves an alteration in protein structure, possibly via an intramolecular thiol disulphide interchange, which influences the interaction of the enzyme with its substrates.
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PMID:Light-dependent de-activation/re-activation of Anabaena variabilis ferredoxin: NADP+ reductase. 190 89

The presence of NADPH-diaphorase enzyme has been previously revealed in fixed mammalian retinal tissue (Sagar, 1986). Fixed retinae of Bufo marinus and Xenopus laevis failed to yield selective staining when reacted for NADPH-diaphorase. Satisfactory staining of retinal neurons was attained when the histochemical reaction was carried out in unfixed retinal wholemounts. The applied method included the following steps: 1) Dissection of the fresh retina and the separation of the neural retina from all other coats of the eye ball, including the vitreal tissue; 2) pretreatment with 300 mM sucrose in phosphate buffer; 3) incubation with NADP, malic acid and nitroblue tetrazolium in phosphate buffer (pH 7.6); and 4) fixation of the tissue in 10% buffered formaldehyde overnight followed by whole mounting. For control, fixed and unfixed rabbit and human retinae were also reacted for NADPH-diaphorase according to the above method. In these species specific staining was achieved only with fixed tissues. The possible implications of revealing NADPH-diaphorase enzyme activity in fixed mammalian and non-fixed anuran tissues are discussed.
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PMID:A method for the demonstration of NADPH-diaphorase activity in anuran species using unfixed retinal wholemounts. 190 62

Pyruvate:NADP+ oxidoreductase from Euglena gracilis, a homodimeric protein with a molecular weight of 309 kDa, is an iron-sulfur flavoenzyme that contains thiamin pyrophosphate (TPP). The functional structure of the enzyme was studied by a limited proteolysis experiment using trypsin. The evidence obtained shows that the enzyme consists of two functional domains, one of which contains an iron-sulfur cluster, which can be isolated as a homodimeric fragment of approximately 220 kDa by proteolysis. The other domain that contains FAD is released as a monomeric fragment of approximately 55 kDa. The pyruvate dehydrogenase reaction is still catalyzed by the large fragment when NADP+ is substituted by methyl viologen, while the small fragment retains a diaphorase-like electron-transfer activity from NADPH to MV. It is thus shown that pyruvate is oxidized in a CoA-dependent reaction to form CO2 and acetyl-CoA in the iron-sulfur domain, and that the two electrons formed are transferred to the FAD domain in which NADP+ is reduced. TPP is considered to be associated in the iron-sulfur domain. The NH2-terminal sequences of the enzyme and its proteolytic fragments reveal that the iron-sulfur domain occurs in the NH2-terminal side of the enzyme. For elucidation of the O2 instability of the enzyme, limited proteolysis was attempted in air. The tryptic fragment derived from the iron-sulfur domain, similar to the native enzyme, appears to be inactivated by direct contact with O2. In contrast, the FAD domain, when separated from the other domain, is quite stable in air, although the diaphorase activity decays when the native enzyme is exposed to O2.
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PMID:Pyruvate:NADP+ oxidoreductase from Euglena gracilis: limited proteolysis of the enzyme with trypsin. 191 Feb 87

The authors studied the cytotoxic function, activity of NAD- and NADP-diaphorases in the peripheral blood lymphocytes in 57 patients with B-cellular variant of chronic lympholeukoses and found a significant reduction of the natural killer activity of lymphocytes, increased activity of NADP-diaphorase. Reduction of natural killer activity in patients with B-cell variant of chronic lympholeukoses did not depend on the activity of membrane diaphorases in peripheral blood lymphocytes.
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PMID:[NAD- and NADP-diaphorase activity in the peripheral blood lymphocytes of patients with the B-cell variant of chronic lympholeukemia]. 204 48

A flavoprotein with properties similar to those of ferredoxin:NADP+ oxidoreductases found in the leaves of higher plants has been purified to apparent homogeneity from bean sprouts, a nonphotosynthetic plant tissue. The absorbance and circular dichroism spectra of the bean sprout protein are similar to those of spinach leaf ferredoxin:NADP+ oxidoreductase and an antibody raised against the spinach enzyme recognized the bean sprout enzyme. The bean sprout enzyme catalyzed ferredoxin-dependent electron transfer from NADPH to equine cytochrome c at a high rate but, unlike the spinach enzyme, exhibited little NADPH to 2,6-dichlorophenol indophenol diaphorase activity. The bean sprout enzyme forms a 1:1 electrostatically stabilized complex with ferredoxins isolated from either bean sprouts or spinach leaves.
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PMID:Characterization of a ferredoxin:NADP+ oxidoreductase from a nonphotosynthetic plant tissue. 210 79

Chemical modification of ferredoxin--NADP+ reductase from the cyanobacteria Anabaena has been performed using the alpha-dicarbonyl reagent phenylglyoxal. Inactivation of both the diaphorase and cytochrome-c reductase activities, characteristic of the enzyme, indicates the involvement of one or more arginyl residues in the catalytic process of the enzyme. The determination of the rate constants for the inactivation process under different conditions, including those in which substrates, NADP+ and ferredoxin, as well as other NADP+ analogs were present, indicates the involvement of two different groups in the inactivation process, one that reacts very rapidly with the reagent (kobs = 8.3 M-1 min-1) and is responsible for the binding of NADP+, and a second less reactive group (kobs = 0.9 M-1 min-1), that is involved in the binding of ferredoxin. Radioactive labeling of the enzyme with [14C]phenylglyoxal confirms that two groups are modified while amino acid analysis of the modified protein indicates that the modified groups are arginine residues. The identification of the amino acid residues involved in binding and catalysis of the substrates of ferredoxin--NADP+ reductase will help to elucidate the mechanism of the reaction catalyzed by this important enzyme.
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PMID:Arginyl groups involved in the binding of Anabaena ferredoxin--NADP+ reductase to NADP+ and to ferredoxin. 210 14

A NADPH cytochrome c oxidoreductase purified from membranes of rabbit peritoneal neutrophil was shown to behave as the NADPH dehydrogenase component of the O2- generating oxidase complex. A photoactivable derivative of NADP+, azido nitrophenyl-gamma-aminobutyryl NADP+ (NAP4-NADP+), was synthesized in its labeled [3H] form and used to photolabel the NADPH cytochrome c reductase at different stages of the purification procedure. Control assays performed in dim light indicated that the reduced form of NADP4-NADP+ generated by reduction with glucose-6-phosphate and glucose-6-phosphate dehydrogenase was oxidized at virtually the same rate as NADPH. Upon photoirradiation of the purified reductase in the presence of [3H]NAP4-NADP+ and subsequent separation of the photolabeled species by sodium dodecyl sulfate polyacrylamide gel electrophoresis, radioactivity was found to be present predominantly in a protein band with a molecular mass of 77-kDa and accessorily in bands of 67-kDa and 57-kDa. Evidence is provided that the 67-kDa and 57-kDa proteins arose from the 77-kDa protein by proteolysis. Despite removal of part of the sequence, the proteolyzed proteins were still active in catalyzing electron transport from NADPH to cytochrome c and in binding the photoactivable derivative of NADP+.
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PMID:Characterization of multiple active forms of the NADPH dehydrogenase component of the oxidase complex from rabbit peritoneal neutrophils by photolabeling with an arylazido derivative of NADP+. 210 11


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