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

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.
 ...
PMID:Pyruvate:NADP+ oxidoreductase from Euglena gracilis: limited proteolysis of the enzyme with trypsin. 191 Feb 87

A method is described for isolating Clara cells from the mouse lung that does not require the technique of elutriation. Mouse lungs totally perfused of blood are instilled with crystalline trypsin (0.25%) and incubated for the optimum time of 15 min. The lung tissue is chopped, mechanically agitated, and sequentially filtered to obtain a primary digest of 3 to 5 x 10(6) cells. Clara cells, identified routinely by histochemical localization of NADPH diaphorase, using the stain nitrotetrazolium blue (NBT), accounts for between 20 to 40% of the cells in the primary digest. Layering the cells of the primary digest on a discontinuous Percoll gradient followed by centrifugation gives rise to a major band of cells, 52% that are Clara cells (0.77 +/- 0.28 x 10(6)/mouse). A second method was devised to purify the Clara cells by simply centrifuging (32g, 6 min, 10 degrees C) the primary digest and discarding the supernatant that contained only a few NBT positive cells. When this process was repeated three times, the final pellet contained 68% Clara cells realizing 0.55 +/- 0.16 x 10(6) cells/mouse. The cells have typical Clara cell morphology as confirmed by electron microscopy and have a high level of P-450 enzymes (7-ethoxycoumarin deethylase and coumarin hydroxylase). Furthermore, the primary digests and the purified isolates contain less than 1% alveolar Type II cells, although such cells, identified by the histochemical localization of alkaline phosphatase, can be obtained by a second, more extensive digestion procedure. The simple procedure described for the isolation of mouse Clara cells could be further advanced if methods could be devised to prevent the loss of NADPH diaphorase activity during enzymatic digestion and cell centrifugation.
 ...
PMID:Isolation of Clara cells from the mouse lung. 220 Jun 69

Ferredoxin-NADP reductase accounts for about 50% of the NADPH diaphorase activity of spinach leaf homogenates. The enzyme is bound to thylakoid membranes, but can be slowly extracted by aqueous buffers. Ferredoxin-NADP reductase can be extracted from the membranes by a 1- to 2-min treatment with a low concentration of trypsin. This treatment completely inactivates NADP photoreduction but does not affect electron transport from water to ferredoxin. It is shown that the inactivation is due to solubilization of ferredoxin-NADP reductase: the activity can be restored by addition of a very large excess of soluble enzyme in pure form. When ferredoxin-NADP reductase is added as a soluble enzyme after extraction or inactivation (by a specific antibody) of the membrane-bound enzyme, NADP photoreduction requires a very large excess of this enzyme, and the apparent Km for ferredoxin is also increased. These observations are discussed as related to the interactions of thylakoids with ferredoxin-NADP reductase.
 ...
PMID:Interaction of ferredoxin and ferredoxin-NADP reductase with thylakoids. 683 5

Aquacobalamin reductase (NADPH), which catalyzes the reduction of aquacobalamin to cob(II)alamin in the synthesis of cobalamin coenzymes, has already been purified from mitochondria of Euglena gracilis and partly characterized. Here, the enzyme was further characterized to clarify its enzymatic properties. The enzyme reduced 2 mol of aquacobalamin per mole of NADPH and had NADPH diaphorase-like activity. The 16 amino acid residues at the NH2-terminal of the enzyme were identical with those of the NADPH diaphorase domain of pyruvate: NADP+ oxidoreductase, which is involved in Euglena wax ester fermentation. Peptide mapping of the aquacobalamin reductase showed that elution during C-18 reversed-phase high-performance liquid chromatography was identical to that of the NADPH diaphorase domain. Immunoblotting indicated that the Euglena aquacobalamin reductase had a higher molecular weight (166,000) in the intact mitochondria than the purified enzyme (65,000), and that the molecular weights of the native and purified enzyme were identical with those of the subunit and the NADPH diaphorase domain, respectively. These results showed that the aquacobalamin reductase isolated earlier was the NADPH diaphorase domain, cleaved by trypsin during preparation of the mitochondrial homogenate from the native enzyme. Purified pyruvate:NADP+ oxidoreductase also had the activity of aquacobalamin reductase, which suggests that the enzyme in Euglena mitochondria has more than one function in the synthesis of cobalamin co-enzymes.
 ...
PMID:Characterization of aquacobalamin reductase (NADPH) from Euglena gracilis. 837 79

We have recently demonstrated that neurotrophins induce reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity in cultured spinal cord neurons. One prominent neuron population of the spinal cord expressing NADPH-diaphorase activity in vivo are preganglionic sympathetic neurons, including those innervating the adrenal medulla. These neurons receive trophic support from their target. We have shown previously that chromaffin cells contain as yet unidentified neurotrophic molecules, which may include releasable factors relevant for the survival and differentiation of developing preganglionic sympathetic neurons. We have studied the influence of proteins derived from bovine chromaffin cells and released by nicotine on NADPH-diaphorase expression in spinal cord cultures established from 16-day-old rat embryos. At this embryonic age, NADPH-diaphorase activity becomes apparent in the spinal cord and predominantly expressed in sympathetic nuclei. Similar to brain-derived neurotrophic factor and neurotrophin-4, a heat- and trypsin-sensitive component from chromaffin cells contained in granule preparations up-regulated the number of NADPH-diaphorase-positive neurons in spinal cord cultures. Combined application of this activity and neurotrophin-4 resulted in an additive effect, indicating that the effect of the chromaffin cell-derived active component is not mediated by one of the trk B ligands. This was confirmed by co-treatment studies with the trk-signalling pathway inhibitor K252b, which did not inhibit the effect of the chromaffin cell-derived protein(s). Further studies revealed that NADPH-diaphorase reactivity is inducible in spinal cord neurons at any time point throughout the entire culture period of six days, suggesting de novo induction of the enzyme rather than a survival-promoting effect of the activity from chromaffin cells. Culture supernatants from nicotine-stimulated bovine chromaffin cells induced NADPH-diaphorase-positive neurons at the same magnitude as the material obtained from chromaffin granule preparations. Our data suggest that chromaffin cell-derived proteins are capable of up-regulating NADPH-diaphorase activity or to induce de novo this transmitter phenotype in neuron populations of the spinal cord, which may include preganglionic sympathetic neurons.
 ...
PMID:A chromaffin cell-derived protein induces the NADPH-diaphorase phenotype in cultured rat spinal cord neurons. 868 18

Phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidic acid (PA) were found to inhibit strongly the citrulline formation activity of neuronal nitric oxide synthase (nNOS; EC 1.14.13.39). Such inhibition was not observed with any other phospholipid examined. A kinetic analysis of purified nNOS showed no significant change in apparent K(m) for L-Arg or NADPH caused by these inhibitory phospholipids. Electron paramagnetic resonance analysis revealed no significant spectral perturbation of the ferriheme or flavin semiquinone upon the addition of PIP2. On the other hand, a lower enhancement of the NADPH diaphorase activity by Ca(2+)-calmodulin was observed in the presence of PIP2 and PA, and the citrulline formation activity was protected from phospholipid inhibition by preincubation with Ca(2+)-calmodulin. Moreover, trypsin digestion analysis showed that the cleavage site within the calmodulin-binding site of nNOS was specifically protected from trypsin by the addition of PIP2 and PA. These results strongly suggest that PIP2 and PA inhibit the citrulline formation activity of nNOS by blocking the interaction of the enzyme with Ca(2+)-calmodulin.
 ...
PMID:Inhibition of neuronal nitric oxide synthase by phosphatidylinositol 4,5-bisphosphate and phosphatidic acid. 1054 74

The NAD(P)H-nitrate reductase complex (overall-NR) of Chlamydomonas reinhardii exhibits two partial activities: NAD(P)H-cytochrome c reductase (diaphorase) and reduced benzyl viologen-NR (terminal-NR). Mild tryptic digestion of the enzyme complex resulted in the loss of both overall and terminal-NR activities, whereas diaphorase activity remained unaltered. The diaphorase activity of mutant 104 and the terminal-NR activity of mutant 305 of C. reinhardii, which are the sole activities related to NR present in these mutants, responded to tryptic treatment to the same extent as the corresponding activities of the wild enzyme complex. Trypsin disassembled the 220-kd NR native complex by destroying the aggregation capability of the diaphorase subunits without affecting their activity nor molecular size (45 kd). A 67-kd thermostable protein, containing molybdenum co-factor, was also released from trypsin-treated NR. This protein lacked diaphorase and NR activities but was able to reconstitute the overall-NR complex by complementation with untreated diaphorase subunit of mutant 104. Our results support a tetrameric structure for the C. reinhardii NR complex, containing two kinds of subunits.
 ...
PMID:Heteromultimeric structure of the nitrate reductase complex of Chlamydomonas reinhardii. 1645 30

A nitrate reductase (EC 1.6.6.1)-inactivating factor has been isolated from 8-day-old wheat leaves. The purification schedule involved ammonium sulfate precipitation, Sephadex G-100 filtration, DEAE-cellulose chromatography, and Sephadex G-150 filtration. No accurate assessment could be made as to the degree of purification relative to crude extract as the inactivating factor could not be detected in crude extract. However a 2,446-fold purification was achieved from the ammonium sulfate fraction to the pooled enzyme from the Sephadex G-150 step.The inactivating factor was heat-labile and had a molecular weight of 37,500. The inactivating factor was particularly sensitive to the divalent metal chelators, 1,10-phenanthroline and bathophenanthroline. Evidence indicated that Fe(2+) may be the functional metal. The trypsin inhibitors N-alpha-p-tosyl-l-lysine chloromethyl ketone and alpha-N-benzoyl-l-arginine were inhibitory. However, phenylmethyl sulfonyl fluoride, an inhibitor of serine peptide hydrolases, was not inhibitory. Neither casein nor hemoglobin nor a range of artificial substrates were hydrolyzed by the inactivating factor. Highly purified wheat leaf nitrite reductase (EC 1.7.99.3) and ribulose 1,5-bisphosphate carboxylase:oxygenase (EC 4.1.1.39) were not affected by the nitrate reductase-inactivating factor.The inactivating factor was more active toward the NADH-nitrate reductase compared to either of the component enzymic activities flavin adenine mononucleotide-nitrate reductase and methyl viologen-nitrate reductase. The NADH-ferricyanide reductase (diaphorase) component was the least sensitive.
 ...
PMID:In Vitro Stability of Nitrate Reductase from Wheat Leaves: III. Isolation and Partial Characterization of a Nitrate Reductase-inactivating Factor. 1666 Oct 24