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)

DNA fragments encoding streptococcal NADH peroxidase (NPXase) have been amplified, cloned and sequenced from the genome of Streptococcus (Enterococcus) faecalis 10C1 (ATCC 11700). The NPXase gene (npr) comprises 1341 base-pairs and is preceded by a typical ribosome binding site. Upstream from the structural gene, putative -10 and -35 promoter regions have been identified, as has a possible factor-independent terminator that occurs in 3'-flanking sequences. The deduced relative molecular mass (Mr = 49,551), amino acid composition and isoelectric point of NPXase are in good agreement with previous values obtained with the purified enzyme. In addition, three sequenced peptides totaling approximately 20% of the protein were located in the npr gene product. From the sequencing data the deduced NPXase sequence shares low but significant homology with the flavoprotein disulfide reductase class of enzymes ranging from 21% for glutathione reductase (GRase) to 28% for thioredoxin reductase. Alignment of NPXase to Escherichia coli GRase allowed the identification of three previously reported fingerprints for the FAD, NADP+ and central domains of GRase, in the peroxidase sequence. In addition, Cys42 of NPXase, which is present as an unusual stabilized cysteine-sulfenic acid in the oxidized enzyme, aligns favorably with the charge-transfer cysteine in E. coli GRase, and both residues closely follow FAD-binding folds found near their respective amino termini. Such sequence characteristics can also be seen in mercuric reductase, lipoamide dehydrogenase and trypanothione reductase, suggesting that all these enzymes may have originally diverged from a common ancestor. Sequences that are on average 50% identical with three previously reported peptides of the related streptococcal NADH oxidase were also identified in the NPXase primary structure, suggesting a strong similarity between these flavoenzymes. Using the E. coli phage T7 expression system the npr gene has now been overexpressed in an E. coli genetic background. The resultant overexpressing clone produced a recombinant NPXase that was catalytically active and immunoreactive to NPXase antisera.
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PMID:Cloning, sequence and overexpression of NADH peroxidase from Streptococcus faecalis 10C1. Structural relationship with the flavoprotein disulfide reductases. 171 12

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

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 glycine-utilizing bacterium Clostridium litoralis contained two enzyme systems for oxidizing dihydrolipoamide. The first one was found to be a genuine dihydrolipoamide dehydrogenase, present only in low amounts. This enzyme had the typical dimeric structure with a subunit molecular mass of about 53 kDa; however, it reacted with both NADP (Km 0.11 mM) and NAD (Km 0.5 mM). The reduction of pyridine nucleotides by dihydrolipoamide was the strongly preferred reaction. A second dihydrolipoamide-oxidizing enzyme system consisted of the interaction of two proteins, the previously described NADP(H)-dependent electron-transferring flavoprotein (D. Dietrichs, M. Meyer, B. Schmidt, and J. R. Andreesen, J. Bacteriol. 172:2088-2095, 1990) and a thioredoxin. This enzyme system was responsible for most of the dihydrolipoamide dehydrogenase activity in cell extracts. The thioredoxin did not bind to DEAE, was heat stable, and had a molecular mass of about 15 kDa. N-terminal amino acid analysis of the first 38 amino acid residues resulted in 38% homology to Escherichia coli thioredoxin and about 76% homology to a corresponding protein isolated from the physiologically close related Eubacterium acidaminophilum. The protein of the latter organism had a molecular mass of about 14 kDa and stimulated the low dihydrolipoamide dehydrogenase activity of the corresponding flavoprotein. By this interaction with NADPH-dependent flavoproteins, a new assay system for thioredoxin was established. A function of thioredoxin in glycine metabolism of some anaerobic bacteria is proposed.
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PMID:Thioredoxin elicits a new dihydrolipoamide dehydrogenase activity by interaction with the electron-transferring flavoprotein in Clostridium litoralis and Eubacterium acidaminophilum. 199 93

The redox behaviour of the NAD(P) system and flavoproteins was registered by simultaneous fluorescence measurements in epididymal bull spermatozoa. The flavoprotein fluorescence signal can nearly exclusively be attributed to an NAD-linked enzyme, alpha-lipoamide dehydrogenase (Em7.4 = -286 mV). A comparison of intact with digitonin-permeabilized spermatozoa revealed that about 50% of the total NAD(P)H fluorescence signal was of mitochondrial origin. Under equilibrium conditions, the midpoint potentials of the NAD(P)H fluorescence signal of both compartments were almost identical (-300 mV). When lactate was present as substrate, 1 mM caffeine increased respiration oxidizing the NAD(P)H system in both mitochondria and cytosol. This indicates a close relationship of the two NAD pools in spermatozoa.
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PMID:Use of NAD(P)H and flavoprotein fluorescence signals to characterize the redox state of pyridine nucleotides in epididymal bull spermatozoa. 200 81

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

Topical application on rat oral mucosa of the chemical 4-nitroquinoline 1-oxide (4NQO) has been shown to produce squamous cell carcinomas on the posterior tongue and/or the posterior hard palate. 4NQO is broken down in vivo by a diaphorase, 4NQO reductase (E.C.1.6.99.2), to produce an active molecule believed to be responsible for carcinogenesis. It has been shown that there are higher concentrations of 4NQO reductase in oesophageal mucosa compared with elsewhere in the gastrointestinal tract. The purpose of these experiments was to compare the distribution of certain diaphorases in the oral mucosa. Samples of rat tongue and cheek epithelia were homogenized, then ultracentrifuged to provide mixed cytosol and microsome fractions from the epithelial cells. A spectrophotometer was used to measure the variation in absorbance at 340 nm of NADH consumed by reduction of 4NQO by enzymes present in the tissue extracts. A histochemical technique was used to compare the activity of NADH diaphorase, NADP diaphorase and glucose-6-phosphate dehydrogenase at different sites of the oral mucosa. Statistical analysis showed that there were significant (P less than 0.01) differences between the activities of all three enzymes at different sites of the oral mucosa. In each case, a higher activity was found at the sites of high incidence of squamous cell carcinoma. A lower activity was found at sites where carcinomas did not occur.
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PMID:A relationship found between intra-oral sites of 4NQO reductase activity and chemical carcinogenesis. 211 96

The 45 kDa diphenylene iodonium-binding flavoprotein of the human neutrophil superoxide-generating oxidase has been purified by affinity chromatography. The polypeptide was eluted from Blue Memsep or 2',5'-ADP-agarose columns with either NADP or low concentrations of the specific inhibitor diphenylene iodonium. The purified protein was shown to bind FAD at a ratio of 1.09 mol of FAD/mol of protein. The reconstituted flavoprotein had a fluorescence spectrum similar, but not identical, to that of free FAD. It had an isoelectric point of approx. 4.0. The reconstituted flavoprotein displayed no diaphorase activity towards a range of artificial electron acceptors. Polyclonal antibodies raised against the pure protein inhibited superoxide generation by solubilized oxidase in a dose-dependent manner, and inhibited superoxide generation when incubated with either cytosol or membrane fractions in a reconstituted system. These antibodies precipitated the 45 kDa polypeptide together with a haem-containing 23 kDa protein thought to be the small subunit of cytochrome b-245. Antibodies raised against cytochrome P-450 reductase also precipitated these two polypeptides. These results are consistent with the 45 kDa polypeptide being the flavoprotein of the neutrophil superoxide-generating oxidase.
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PMID:Purification and some properties of the 45 kDa diphenylene iodonium-binding flavoprotein of neutrophil NADPH oxidase. 215 84


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