Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P30044 (antioxidant enzyme)
 8,037 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The activity of pure calf-liver and Escherichia coli thioredoxin reductases decreased drastically in the presence of NADPH or NADH, while NADP+, NAD+ and oxidized E. coli thioredoxin activated both enzymes significantly, particularly the bacterial one. The loss of activity under reducing conditions was time-dependent, thus suggesting an inactivation process: in the presence of 0.24 mM NADPH the half-lives for the E. coli and calf-liver enzymes were 13.5 and 2 min, respectively. Oxidized E. coli thioredoxin fully protected both enzymes from inactivation, and also promoted their complete reactivation after only 30 min incubation at 30 degrees C. Lower but significant protection and reactivation was also observed with NADP+ and NAD+. EDTA protected thioredoxin reductase from NADPH inactivation to a great degree, thus indicating the participation of metals in the process; EGTA did not protect the enzyme from redox inactivation. Thioredoxin reductase was extensively inactivated by NADPH under aerobic and anaerobic conditions, thus excluding the participation of O2 or oxygen active species in redox inactivation. The loss of thioredoxin reductase activity promoted by NADPH was much faster and complete in the presence of NAD+ glycohydrolase, thus suggesting that inactivation was related to full reduction of the redox-active disulfide. Those results indicate that thioredoxin reductase activity can be modulated in bacteria and mammals by the redox status of NADP(H) and thioredoxin pools, in a similar way to glutathione reductase. This would considerably expand the regulatory potential of the thioredoxin-thioredoxin reductase system with the enzyme being self-regulated by its own substrate, a regulatory protein.
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PMID:NADPH and oxidized thioredoxin mediate redox interconversion of calf-liver and Escherichia coli thioredoxin reductase. 131 49

Inhibition of neurotransmitter release by tetanus toxin and botulinum neurotoxin A can be mimicked by intracellular application of the corresponding toxin light chains. The aim of this study was to determine whether the two-chain toxins are reduced by brain preparations to yield free light chains which would represent the ultimate toxins. The interchain disulfide of two-chain tetanus toxin was cleaved by rat cortex homogenate fortified with NADPH. Reduction was promoted further by addition of thioredoxin. Thioredoxin reductase was demonstrated in and purified from porcine brain cortex. The thioredoxin system which consisted of purified enzyme, thioredoxin and NADPH reduced both toxins. The resulting light chains appeared homogeneous in SDS gel electrophoresis. The complementary heavy chain of tetanus but not of botulinum toxin migrated in two bands, the faster one with the velocity of heavy chain obtained by chemical reduction. The major, slower form was converted into the faster by chemical but not by enzymatic reduction. Tetanus toxin, whether in its single-chain or two-chain version also occurred in two forms which differed by their electrophoretic mobility. The two forms of single-chain toxin were interconverted by chemical reduction or oxidation but not by the thioredoxin system. It is concluded that a) a thioredoxin system in brain tissue reduces the interchain disulfide of two-chain tetanus toxin and botulinum neurotoxin A, b) tetanus toxin but not botulinum neurotoxin A consists of two electrophoretically distinct forms which differ by the thiol-disulfide status of their heavy chains, c) the disulfide loop within the heavy chain of tetanus toxin is resistant to the thioredoxin system.
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PMID:Reductive cleavage of tetanus toxin and botulinum neurotoxin A by the thioredoxin system from brain. Evidence for two redox isomers of tetanus toxin. 157 25

The importance of thioproteins, essential to the ribonucleotide reduction pathway, has been demonstrated in human primary and metastatic melanoma tissues. The thioredoxin reductase/thioredoxin and the glutathione reductase/glutathione/glutaredoxin electron transfer pathways represent alternative electron donors for ribonucleotide reductase and regulate the synthesis of deoxyribonucleotides, the substrates for DNA synthesis, in the S phase of the cell cycle. In addition to their important role in DNA synthesis and cell division, these thioproteins provide effective antioxidant defence against oxygen radicals and hydrogen peroxide. In human metastatic melanoma cells and tissues the thioredoxin reductase/thioredoxin system is located both in the cell cytosol and on plasma membranes and is under allosteric regulation by calcium. As a consequence, calcium plays an important role in determining the intracellular redox status, cell division and differentiation. Recently, the intracellular redox conditions have been shown to be important in the reaction of alkylating anti-tumour drugs such as the chloroethylnitrosoureas. In addition to previously established mechanisms, these highly reactive drugs inhibit thioredoxin reductase, glutathione reductase and ribonucleotide reductase by chloroethylation of their respective thiolate active sites. Incorporation of the 14C chloroethyl group in drug sensitive and resistant human metastatic melanoma cell lines depends on the redox status, with resistant cells being more oxic than sensitive cells. Thioredoxin reductase is 500-fold more sensitive than glutathione reductase to the newly developed nitrosourea, Fotemustine (diethyl-1-[3,2 chloroethyl]-3-nitrosoureido ethyl phosphonate). It has been shown that melanomas which respond to Fotemustine therapy contain more thioredoxin reductase whereas resistant metastases yielded the opposite result.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:New aspects in the pathophysiology of cutaneous melanoma: a review of the role of thioproteins and the effect of nitrosoureas. 184 12

Thioredoxin reductase (TR) is a widely distributed flavoenzyme that provides reduced thioredoxin, a dithiol hydrogen donor for protein disulfide reduction and for the reduction of ribonucleotides to deoxyribonucleotides, the first unique step of DNA synthesis. Antitumor quinones were found to exhibit time- and concentration-dependent inhibition of purified rat liver TR that requires the presence of NADPH. Diaziquone initially shows competitive inhibition of the enzyme with 5,5'-dithiobis 2-nitrobenzoic acid as substrate with a Ki of 7.5 microM, which becomes non-competitive after 1 hour incubation with NADPH with a Ki of 0.5 microM. Doxorubicin shows non-competitive inhibition both initially and after 1 hr incubation with NADPH, with Ki values of 10 microM and 0.5 microM, respectively. Electron spin resonance spectroscopy showed the formation of semiquinone free radicals by TR incubated under anaerobic conditions with doxorubicin or diaziquone and NADPH. Redox cycling and formation of oxygen radicals does not play a major role in the inhibition of TR by antitumor quinones as shown by the minor effect on inhibition of removing O2, and the lack of effect of superoxide dismutase and catalase. Diaziquone causes time- and concentration-dependent inhibition of TR activity in intact A204 human rhabdomyosarcoma cells that is associated with growth inhibition. The results suggest that inhibition of TR by antitumor quinones could contribute to their growth inhibitory properties.
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PMID:Inhibition of thioredoxin reductase (E.C. 1.6.4.5.) by antitumor quinones. 216 13

Scavenging mechanisms for persistent free radicals were investigated using nitroxide-type radicals as model compounds. The free radical reducing activity of a) isolated thioredoxin reductase, a flavin containing oxidoreductase, b) skin homogenates, and c) the epidermis of hairless mice was studied by electron spin resonance spectroscopy. In all three systems, reduction rates of different classes of nitroxide free radicals exhibited the following order: oxazolidinoxy greater than piperidinoxy greater than dihydropyrroloxy. The main reductant for piperidinoxy radicals in mouse skin homogenate is ascorbic acid. Other reducing activities were stimulated by NAD(P)H and could be inhibited by N-ethyl maleimide, suggesting involvement of thiol-dependent processes. Mammalian thioredoxin, a competitive inhibitor of nitroxide reduction by thioredoxin reductase, significantly stimulates nitroxide scavenging in skin homogenate. Thioredoxin reductase did not significantly participate in nitroxide reduction in skin homogenates. At the surface of mouse epidermis a cationic dihydropyrroloxy nitroxide, which was stable in the presence of mammalian thioredoxin reductase was readily reduced. The epidermal reduction was inhibited by zinc, N-ethyl maleimide, and by heat (70 degrees C, 5 min). At least for mouse epidermis, reduction of a variety of nitroxides is a complex phenomenon involving enzymatic and nonenzymatic mechanisms and cannot be used as a specific assay for an enzyme, e.g., thioredoxin reductase. The study indicates the epidermis contains an effective antioxidant system that scavenges ascorbate-sensitive piperidinoxy nitroxides as well as more reducing radicals exemplified by dihydropyrroloxy nitroxides.
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PMID:Free radical reduction mechanisms in mouse epidermis skin homogenates. 238 May 82

Thioredoxin reductase (TRR), a member of the pyridine nucleotide-disulfide oxidoreductase family of flavoenzymes, undergoes two sequential thiol-disulfide interchange reactions with thioredoxin during catalysis. In order to assess the catalytic role of each nascent thiol of the active site disulfide of thioredoxin reductase, the 2 cysteines (Cys-136 and Cys-139) forming this disulfide have been individually changed to serines by site-directed mutageneses of the cloned trxB gene of Escherichia coli. Spectral analyses of TRR(Ser-136,Cys-139) as a function of pH and ionic strength have revealed two pKa values associated with the epsilon 456, one of which increases from 7.0 to 8.3 as the ionic strength is increased, and a second at 4.4 which is seen only at high ionic strength. epsilon 458 of wild type TRR(Cys-136,Cys-139) and epsilon 453 of TRR(Cys-136,Ser-139) are pH-independent. A charge transfer complex (epsilon 530 = 1300 M-1 cm-1), unique to TRR(Ser-136,Cys-139), has been observed under conditions of high ammonium cation concentration (apparent Kd = 54 microM) at pH 7.6. These results suggest the assignment of Cys-139 as the FAD-interacting thiol in the reduction of thioredoxin by NADPH via thioredoxin reductase. If, as with other members of this enzyme family, the two distinct catalytic functions are each carried out by a different nascent thiol, then Cys-136 would perform the initial thiol-disulfide interchange with thioredoxin. Steady state kinetic analyses of the proteins have revealed turnover numbers of 10 and 50% of the value of the wild type enzyme for TRR(Ser-136,Cys-139) and TRR(Cys-136,Ser-139), respectively, and no changes in the apparent Km values of TR(S2) or NADPH. The finding of activity in the mutants indicates that the remaining thiol can carry out interchange with the disulfide of thioredoxin, and the resulting mixed disulfide can be reduced by NADPH via the flavin.
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PMID:Characterization of two active site mutations of thioredoxin reductase from Escherichia coli. 264 68

Thioredoxin reductase has been purified from human metastatic melanotic melanoma and amelanotic melanoma tissues. Enzyme from the melanotic melanoma tissue contains bound calcium showing classical sigmoidal allosteric kinetics, whereas enzyme from the amelanotic melanoma yielded normal Michaelis-Menten saturation with substrate. Calcium inhibition can be partially reversed by oxidized thioredoxin. 45Ca has been used to label the amelanotic melanoma enzyme in order to determine the number of calcium-binding sites. These isotope experiments yielded only one calcium-binding site per enzyme molecule. Enzyme labeled with 45Ca was dialyzed for 24 h without loss of radioactivity, but the addition of oxidized thioredoxin to this labeled enzyme caused 60% calcium exchange in 24 h. Comparative studies with Escherichia coli thioredoxin reductase showed similar calcium inhibition as well as partial reactivation with oxidized thioredoxin. The enzyme from E. coli previously sequenced by others, showed considerable homology with the first EF-hands calcium-binding site of calmodulin. Detailed calcium-binding studies indicated that 10(-5) M of this fast exchange ion was sufficient to cause allosteric regulation in 10 min. This strong calcium-binding property could explain the allosteric nature of the thioredoxin reductase purified from human metastatic melanotic melanoma and its role in the regulation of melanin biosynthesis.
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PMID:Calcium regulates thioredoxin reductase in human metastatic melanoma. 276 62

T7 DNA polymerase reduced insulin at the same Km as thioredoxin, while the turnover number decreased. Recycling of the disulfide of thioredoxin subunit to its dithiol form was made by thioredoxin reductase. Incubation of T7 DNA polymerase with insulin decreases its ability to bind DNA and therefore inhibited polymerase and exonuclease activities. Thioredoxin reductase fully reversed this inhibition. Insulin did not induce dissociation of the T7 DNA polymerase subunits, which was tested by immunoadsorbent chromatography. No significant difference in single-stranded exonuclease compared to polymerase activity was seen in the flow through or the eluate, which had been expected if a dissociation of the subunits had occurred.
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PMID:Inhibition of the T7 DNA polymerase by insulin. 676 Nov 43

A reproducible scheme has been developed for the preparation of rat liver thioredoxin and thioredoxin reductase (EC 1.6.4.5) by using assays based on reduction of insulin and 5,5'-dithiobis(2-nitrobenzoic acid), respectively. Both proteins were purified to homogeneity, as judged from polyacrylamide gel electrophoresis. Thioredoxin had a molecular weight of 12 000 and contained about 110 amino acids including 4 half-cystines and an NH2-terminal valine. Peptide maps of reduced and carboxymethylated thioredoxin showed that the protein had the active center sequence -Cys-Gly-Pro-Cys-Lys-Met- characteristic of thioredoxins also from procaryotes. Prolonged air oxidation of fully reduced thioredoxin created inactive, aggregated disulfide-containing molecules. Thioredoxin reductase showed a subunit molecular weight of 58 000 and a native molecular weight of 116 000. The enzyme was highly specific for NADPH with a Km of 6 microM. It contained FAD as prosthetic group and was sensitive to inhibition by arsenite. Thioredoxin reductase had a Km of 2.5 microM for rat and calf liver thioredoxin and a Kcat of 3000 min-1.
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PMID:Rat liver thioredoxin and thioredoxin reductase: purification and characterization. 715 51

Three different molecular masses (24, 22, and 20 kDa) of antioxidant proteins were purified in Escherichia coli. These proteins exhibited the preventive effects against the inactivation of glutamine synthetase activity and the cleavage of DNA by a metal-catalyzed oxidation system capable of generating reactive oxygen species. Their antioxidant activities were supported by a thiol-reducing equivalent such as dithiothreitol. Analysis of the amino-terminal amino acid sequences and the immunoblots between 24- and 22-kDa proteins indicates that the 24-kDa protein is an intact form of the 22-kDa protein that was previously identified 22-kDa subunit (AhpC) of E. coli alkyl hydroperoxide reductase (AhpC/AhpF). We isolated and sequenced an E. coli genomic DNA fragment that encodes 20-kDa protein. Comparison of the deduced amino acid sequence of the 20-kDa protein with that of AhpC revealed no sequence homology. A search of a data bank showed that the 20-kDa protein is a new type of antioxidant enzyme. The synthesis of this novel 20-kDa protein was increased in response to oxygen stress during growth. The 20-kDa protein resides mainly in the periplasmic space of E. coli, whereas the 24-kDa AhpC resides mainly in the matrix. The 20-kDa protein was functionally linked to the thioredoxin as an in vivo thiol-regenerating system and exerted a peroxidase activity. This 20-kDa protein is thus named "thiol peroxidase," which could act as an antioxidant enzyme removing peroxides or H2O2 within the catalase- and peroxidase-deficient periplasmic space of E. coli.
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PMID:Thioredoxin-linked "thiol peroxidase" from periplasmic space of Escherichia coli. 749 81


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