Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q8NEX9 (reductase)
 26,410 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Rat hepatic microsomal squalene synthetase (EC 2.5.1.21) was induced 25-fold by feeding rats with diet containing the hydroxymethylglutaryl-coenzyme A reductase inhibitor, fluvastatin, and cholestyramine, a bile acid sequestrant. A soluble squalene synthetase protein with an estimated mass of 32-35 kDa, as determined by gel filtration chromatography on Sephacryl S-200 column, was solubilized out of the microsomes by controlled proteolysis with trypsin. Approximately 25% of the activity was recovered in a soluble form. The enzyme was purified to homogeneity utilizing a series of column chromatography purification steps on DEAE-cellulose, hydroxylapatite, and phenyl-Sepharose sequentially. The purified enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Initial kinetic analysis indicated an S0.5 values for trans-farnesyl diphosphate of 1.0 microM and for NADPH of 40 microM. The Vmax with respect to trans-farnesyl diphosphate was calculated at 1.2 mumol/min/mg. NADH also serves as substrate for the reaction with S0.5 value of 800 microM. Western blot analysis utilizing rabbit antisera raised against the purified, trypsin-truncated enzyme showed a single band for the isolated solubilized enzyme at 32-33 kDa and a band for the intact microsomal enzyme at about 45-47 kDa.
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PMID:Solubilization, purification, and characterization of a truncated form of rat hepatic squalene synthetase. 156 7

5,10-Methylenetetrahydromethanopterin dehydrogenase and 5,10-methylenetetrahydromethanopterin reductase have been purified to homogeneity by a factor of 86 and 68, respectively, from methanol-grown Methanosarcina barkeri cells. The dehydrogenase was isolated as a hexamer of a single 35 kDa subunit, whereas the reductase was composed of four identical 38 kDa subunits. The purified oxygen-stable enzymes catalyzed the oxidation of 5,10-methylenetetrahydromethanopterin and methyltetrahydromethanopterin with Vmax values of 3000 and 200 mumol min-1 mg-1, respectively. The methanogenic electron carrier coenzyme F420 was a specific electron acceptor for both enzymes. Steady state kinetics for the two enzymes were in agreement with ternary complex (sequential) mechanisms. Methylene reductase and methylene dehydrogenase are proposed to function in the methanol oxidation step to CO2.
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PMID:Purification and properties of 5,10-methylenetetrahydromethanopterin dehydrogenase and 5,10-methylenetetrahydromethanopterin reductase, two coenzyme F420-dependent enzymes, from Methanosarcina barkeri. 191 53

Ferredoxin-NADP reductase (FNR) was rapidly isolated from spinach leaves with special care to suppress proteolytic degradation. The molecular mass of this FNR preparation was estimated to be 35 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Limited proteolysis of 35-kDa FNR to 33-kDa FNR was effectively suppressed by high pH (at pH 9.3), concentrated salts, and low temperature. On the basis of these observations, a new isolation procedure was designed to obtain 35-kDa FNR in a preparative scale. The resulting final preparation still contained two FNR components. One appeared to correspond to the longest polypeptide so far reported for spinach FNR (Karplus et al., 1984, Biochemistry 23, 6576-6583) while the other lacked a gamma-pyroglutamyl residue from its amino terminus. Conventional preparation procedure without suppression of proteolytic action yielded an FNR preparation with a molecular mass of 33 kDa. This FNR preparation consisted of three components. They lacked 11 to 17 amino-terminal residues, while their carboxyl-terminal structure was retained intact. These results showed that proteolytic degradation of the spinach FNR molecule during purification took place exclusively at its amino-terminal moiety and further suggested that 35-kDa FNR with Karplus' structure should be the mature FNR molecule functional in the chloroplast thylakoids.
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PMID:Proteolytic degradation of ferredoxin-NADP reductase during purification from spinach. 218 5

A cDNA clone for the preprotein of spinach ferredoxin:NADP+ reductase has been modified to allow the expression in Escherichia coli of the mature flavoprotein form the lacks the transit peptide. An expression vector, pFNR1, was constructed by subcloning the fragment into the plasmid pDS12/RBSII, SphI. In the crude extracts of transformed cells after induction, two active holoproteins of 35 kDa and 32 kDa, respectively, were found. The 32-kDa protein, purified by immunoaffinity chromatography, was found to lack the first 28 residues of the spinach protein sequence and to have a methionine as the N-terminal residue instead of Val29. A new expression plasmid, pFNR2, was obtained by in vitro mutagenesis of the codon GTG for Val29 to the synonymous GTT; in this case, only the 35-kDa protein was expressed by transformed cells. Both the 35-kDa and 32-kDa enzymes were purified and characterized. All the properties analyzed of the cloned 35-kDa enzyme were very similar to those of the spinach flavoprotein. The 32-kDa form showed the same catalytic efficiency of the spinach enzyme as a diaphorase but its interaction with oxidized ferredoxin was partially impaired.
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PMID:Expression in Escherichia coli of ferredoxin:NADP+ reductase from spinach. Bacterial synthesis of the holoflavoprotein and of an active enzyme form lacking the first 28 amino acid residues of the sequence. 220 97

Membrane-bound fatty acyl-CoA reductase from the green alga Botryococcus braunii has been solubilized from the microsomal preparation by 0.1% octyl beta-glucoside and purified to near homogeneity by Blue A agarose and palmitoyl-CoA agarose affinity column chromatography. The molecular mass of the enzyme was estimated by SDS-PAGE to be 35 kDa. The enzyme generates fatty aldehyde by reduction of fatty acyl-CoA with NADH as the reductant. The N-terminal amino acid sequence of this protein that represents the first eucaryotic aldehyde-generating reductase to be purified shows high homology with the N-terminus of fatty acid reductase from bacteria.
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PMID:Solubilization and purification of aldehyde-generating fatty acyl-CoA reductase from green alga Botryococcus braunii. 764 95

Maleylacetate reductase (EC 1.3.1.32) plays a major role in the degradation of chloroaromatic compounds by channeling maleylacetate and some of its substituted derivatives into the 3-oxoadipate pathway. The enzyme was purified to apparent homogeneity from an extract of 2,4-dichlorophenoxyacetate (2,4-D)-grown cells of Alcaligenes eutrophus JMP134. Maleylacetate reductase appears to be a dimer of two identical subunits of 35 kDa. The pI was determined to be at pH 5.4. There was no indication of a flavin prosthetic group. The enzyme was inactivated by p-chloromercuribenzoate but not by EDTA, 1,10-phenanthroline, or dithiothreitol. Maleylacetate and 2-chloromaleylacetate were converted with similar efficiencies (with NADH as cosubstrate, Km = 31 microM for each substrate and kcat = 8,785 and 7,280/min, respectively). NADH was preferred to NADPH as the cosubstrate. Upon reduction of 2-chloramaleylacetate by the purified enzyme, chloride was liberated and the resulting maleylacetate was further reduced by a second NADH. These results and the kinetic parameters suggest that the maleylacetate reductase is sufficient to channel the 2,4-D degradation intermediate 2-chloromaleylacetate into the 3-oxoadipate pathway. In a data base search the NH2-terminal sequence of maleylacetate reductase was found to be most similar to that of TfdF, a pJP4-encoded protein of as-yet-unknown function in 2,4-D degradation.
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PMID:Purification and characterization of maleylacetate reductase from Alcaligenes eutrophus JMP134(pJP4). 822 15

Proteins of the peroxisomal membrane can be schematically divided into two groups, one being made up of more or less characterized proteins with generally unknown functions and the other consisting of enzyme activities of which the corresponding proteins have not been characterized. In the present report, these proteins and enzymes are described with the addition of unpublished results regarding their induction by peroxisome proliferators at the post-transcriptional level. Integral membrane proteins (IMPs) can be isolated using an alkaline solution of sodium carbonate. A dozen of preponderant IMPs can be seen on sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the major band corresponds to a 70 kDa IMP, of which the corresponding rat cDNA is known. Some IMPs have been characterized by immunoblot analysis. Recently, a cDNA has been cloned for a peroxisome assembly factor (35 kDa IMP). Functions have also been proposed for some IMPs but are not yet firmly settled. Some IMPs (450/520, 70 and 26 kDa) are strongly induced by peroxisome proliferators. Our results extend to cipro- and fenofibrate the observation that the 70 kDa IMP mRNA level is strongly increased in di(2-ethylhexyl)phtalate-treated rats. All the enzyme activities associated with the peroxisomal membrane are involved in lipid metabolism: activation of substrates (fatty acids), ether lipid biosynthesis, and formation of precursors (fatty alcohols). It is believed that the same long-chain acyl-CoA synthetase occurs in the peroxisome as well as in the outer mitochondrial membrane and the endoplasmic reticulum. However, two highly homologous but different cDNAs encoding rat liver and brain long-chain acyl-CoA synthetases have been isolated recently. Evidence has been accumulated for a distinct synthetase that specifically activates very-long chain fatty acids. The first two steps of ether lipid biosynthesis require dihydroxyacetone-phosphate (DHAP) acyltransferase and alkyl-DHAP synthetase, the active sites of which are located on the inner surface of the membrane. In contrast, the catalytic site of the acyl/alkyl-DHAP reductase, which generates sn-1-alkyl-glycerol-3-phosphate, is located on the outer surface. Long-chain fatty alcohols, which are obligate precursors of ether lipids and wax esters, are biosynthetized by the reduction of the corresponding acyl-CoAs via the action of an acyl-CoA reductase. Peroxisome proliferators do not appear to stimulate these enzyme activities specifically. However, we report that feno- and ciprofibrate treatments increase six-fold the palmitoyl-CoA synthetase mRNA level in the rat liver.
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PMID:Proteins and enzymes of the peroxisomal membrane in mammals. 851 48

We describe the purification and characterisation of a thioredoxin reductase-like disulphide reductase from the ancient protozoan parasite, Giardia duodenalis. This dimeric flavoprotein contains 1 mol FAD per subunit and had an apparent subunit molecular mass of 35 kDa. The purified enzyme catalysed the NADPH-dependent (Km = 8 microM) reduction of 5,5'-dithio-bis(2-nitrobenzoic acid) to thionitrobenzoate and was unable to utilise NADH as an electron donor. The sulphydryl-active compounds, N-ethylmaleimide, sodium arsenite and Zn2+ ions, strongly inhibited the enzyme suggesting that a thiol component forms part of the active site. Purified enzyme was able to utilise a variety of substrates, including cystine and oxidised glutathione, which suggests that it is a broad-range disulphide reductase, probably accounting for the majority of thiol cycling activity in this organism. While the G. duodenalis enzyme does not require an intermediate electron transport protein, analogous to thioredoxin, for activity, we have identified a candidate carrier protein which enhances DTNB turnover six fold, therefore implying that Giardia contains a thioredoxin-like system. Physical, enzymatic and spectral properties of the G. duodenalis disulphide reductase are also consistent with it being a member of the thioredoxin reductase-class of disulphide reductases. Furthermore, the internal amino acid sequence of a tryptic peptide generated from the purified protein was highly homologous with thioredoxin reductases from other sources. This is the first report of a disulphide reductase to be purified from the anaerobic protozoa and explains the so called "glutathione-induced thiol-reductase activity' previously observed in G. duodenalis.
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PMID:A thioredoxin reductase-class of disulphide reductase in the protozoan parasite Giardia duodenalis. 902 54

A protein detected in N-methyl-N-nitrosourea-initiated rat hepatomas by two-dimensional electrophoresis at 35 kDa/pI 7.4 was identified in a previous study by internal amino acid micro sequencing as an aldose-reductase-like protein [Zeindl-Eberhart, E., Jungblut, P. R., Otto, A. & Rabes, H. M. (1994) Identification of tumor-associated protein variants during rat hepatocarcinogenesis, J. Biol. Chem. 269, 14589-14594]. Two-dimensional electrophoresis of rat lens proteins revealed a spot at 37 kDa/pI 6.8 that showed a high degree of identity (98.5%) with rat lens aldose reductase after amino acid sequencing and 80% sequence identity to the rat-hepatoma-derived aldose-reductase-like protein. This suggests that hepatoma-derived aldose-reductase-like protein and rat lens aldose reductase are related proteins encoded by different genes. A different expression profile of these proteins was found in various rat organs. Rat lens aldose reductase is present, in addition to in lens, in heart, brain, muscle, lung, duodenum, kidney, spleen and bone marrow, while the hepatoma-derived aldose-reductase-like protein is found preferentially in hepatomas and in embryonic liver. Though different in organ expression, an identical response was found for both proteins after stimulation with fibroblast growth factor-1 and after exposure to increased glucose concentrations. Since rat hepatoma-derived aldose-reductase-like protein is expressed in embryonic, but not in adult liver, it is assumed that it is expressed in hepatomas as a functionally active embryonal type of aldose reductase during hepatocarcinogenesis. Immunohistochemistry revealed that the hepatoma-derived aldose-reductase-like protein is expressed already in the preneoplastic stage of hepatocarcinogenesis and might potentially serve as a marker enzyme in early hepatic neoplasia.
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PMID:Further characterization of a rat hepatoma-derived aldose-reductase-like protein--organ distribution and modulation in vitro. 928 99

The 29 kDa protein of Entamoeba histolytica (Eh29), as well as a truncated variant of this protein, which lacks a cysteine-rich N-terminal region of 40 amino acid residues (Eh29mut), were recombinantly expressed in Escherichia coli and purified to homogeneity. Both recombinant proteins (recEh29, recEh29mut) were found to have hydrogen peroxide (H2O2)-removing activity, but recEh29 was twice as active as recEh29mut. For the consumption of exogenous H2O2, activity was dependent on the presence of reducing equivalents, such as dithiothreitol (DTT), indicating that Eh29 constitutes a thiol-dependent peroxidase. DTT was not required to remove H2O2 by recEh29 or recEh29mut when H2O2 was generated enzymically by the E. histolytica NADPH:flavin oxidoreductase. This enzyme produces H2O2 under aerobic conditions and simultaneously serves as a hydrogen donor for Eh29. Peroxidase activity of the recombinant proteins was further supported by complementation of an E. coli strain that lacks the entire alkyl hydroperoxide reductase locus. The high sensitivity of these bacteria against cumene hydroperoxide was significantly reduced by the introduction of the genes encoding recEh29 or recEh29mut. Using antisera raised against the recombinant proteins, native Eh29 was localized within the cytoplasm of the amoebae. In addition, the antisera reacted with proteins of E. histolytica lysates with apparent molecular masses of 35 kDa and 160-300 kDa. All of them exhibited thiol-peroxidase activity.
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PMID:Removal of hydrogen peroxide by the 29 kDa protein of Entamoeba histolytica. 930 28


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