Binding of ATP and its derivatives to selenophosphate synthetase from Escherichia coli.

Mechanistically similar selenophosphate synthetases (SPS) have been isolated from different organisms. SPS from Escherichia coli is an ATP-dependent enzyme with a C-terminal glycine-rich Walker sequence that has been assumed to take part in the first step of ATP binding. Three C-terminally truncated mutants of SPS, containing the N-terminal 238 (SPS(238)), 262 (SPS(262)), and 332 (SPS(332)) amino acids of the 348-amino-acid protein, have been extracted from cell pellets, and two of these (SPS(262) and SPS(332)) have been purified to homogeneity. SPS(238) has been obtained in a highly purified form. Binding of the fluorescent ATP-derivative TNP-ATP and Mn-ATP to the proteins was examined for all truncated mutants of SPS and a catalytically inactive C17S mutant. It has been shown that TNP-ATP can be used as a structural probe for ATP-binding sites of SPS. We observed two TNP-ATP binding sites per molecule of enzyme for wild-type SPS and SPS(332) mutant and one TNP-ATP binding site for SPS(238) mutant. The stoichiometry of Mn-ATP-binding was 2 mol of ATP per mol of protein determined with [(14)C]ATP by HPLC gel-filtration column chromatography under saturating conditions. The binding stoichiometries for SPS(332), SPS(262), and SPS(238) were 2, 1.6, and 1, respectively. The C17S mutant exhibits about one third of wild type SPS TNP-ATP-binding ability and converts 12% of ATP in the ATPase reaction to ADP in the absence of selenide. The C-terminus contributes two thirds to the TNP-ATP binding; SPS(238) likely has one ATP-binding site removed by truncation.

Cloning and heterologous expression of a Methanococcus vannielii gene encoding a selenium-binding protein.

The activation and incorporation of selenium into selenocysteine containing selenoproteins has been well established in an Escherichia coli model system but there is little specific information concerning the transport and intracellular trafficking of selenium in biological systems in general. A selenium transport role is a possible function of a novel 42 kDa selenium-binding protein that recently was purified from Methanococcus vannielii. The gene encoding a monomer of this protein (Sbp) has been cloned, sequenced and heterologously expressed in E. coli. The 8.8 kDa gene product contains 81 amino acids. The recombinant Sbp (rSbp) protein was shown to bind selenium from added selenite. The bound selenium appeared predominantly in dimeric and tetrameric forms of the protein. The gene encoding Sbp occurs in an operon that contains a carbonic anhydrase gene and selenocysteine-containing formate dehydrogenase genes, suggesting possible roles in selenium-dependent formate metabolism.

Utilization of selenocysteine as a source of selenium for selenophosphate biosynthesis.

Selenophosphate synthetase (SPS), the selD gene product from Escherichia coli, catalyzes the biosynthesis of monoselenophosphate from selenide and ATP. Characterization of selenophosphate synthetase revealed the determined K(m) value for selenide is far above the optimal concentration needed for growth and approached levels which are toxic. Selenocysteine lyase enzymes, which decompose selenocysteine to elemental selenium (Se(0)) and alanine, were considered as candidates for the control of free selenium levels in vivo. The ability of a lyase protein to generate Se(0) in the proximity of SPS maybe an attractive solution to selenium toxicity as well as the high K(m) value for selenide. Recently, three E. coli NifS-like proteins, CsdB, CSD, and IscS, were characterized. All three proteins exhibit lyase activity on L-cysteine and L-selenocysteine and produce sulfane sulfur, S(0), or Se(0) respectively. Each lyase can effectively mobilize Se(0) from L-selenocysteine for selenophosphate biosynthesis.

Formation of a selenium-substituted rhodanese by reaction with selenite and glutathione: possible role of a protein perselenide in a selenium delivery system.

Selenophosphate is the active selenium-donor compound required by bacteria and mammals for the specific synthesis of Secys-tRNA, the precursor of selenocysteine in selenoenzymes. Although free selenide can be used in vitro for the synthesis of selenophosphate, the actual physiological selenium substrate has not been identified. Rhodanese (EC ) normally occurs as a persulfide of a critical cysteine residue and is believed to function as a sulfur-delivery protein. Also, it has been demonstrated that a selenium-substituted rhodanese (E-Se form) can exist in vitro. In this study, we have prepared and characterized an E-Se rhodanese. Persulfide-free bovine-liver rhodanese (E form) did not react with SeO(3)(2-) directly, but in the presence of reduced glutathione (GSH) and SeO(3)(2-) E-Se rhodanese was generated. These results indicate that the intermediates produced from the reaction of GSH with SeO(3)(2-) are required for the formation of a selenium-substituted rhodanese. E-Se rhodanese was stable in the presence of excess GSH at neutral pH at 37 degrees C. E-Se rhodanese could effectively replace the high concentrations of selenide normally used in the selenophosphate synthetase in vitro assay in which the selenium-dependent hydrolysis of ATP is measured. These results show that a selenium-bound rhodanese could be used as the selenium donor in the in vitro selenophosphate synthetase assay.

Overexpression of wild type and SeCys/Cys mutant of human thioredoxin reductase in E. coli: the role of selenocysteine in the catalytic activity.

In contrast to Escherichia coli and yeast thioredoxin reductases, the human placental enzyme contains an additional redox center consisting of a cysteine-selenocysteine pair that precedes the C-terminal glycine residue. This reactive selenocysteine-containing center imbues the enzyme with its unusually wide substrate specificity. For expression of the human gene in E. coli, the sequence corresponding to the SECIS element required for selenocysteine insertion in E. coli formate dehydrogenase H was inserted downstream of the TGA codon in the human thioredoxin reductase gene. Omission of this SECIS element from another construct resulted in termination at UGA. Change of the TGA codon to TGT gave a mutant enzyme form in which selenocysteine was replaced with cysteine. The three gene products were purified using a standard isolation protocol. Binding properties of the three proteins to the affinity resins used for purification and to NADPH were similar. The three proteins occurred as dimers in the native state and exhibited characteristic thiolate-flavin charge transfer spectra upon reduction. With DTNB as substrate, compared to native rat liver thioredoxin reductase, catalytic activities were 16% for the recombinant wild type enzyme, about 5% for the cysteine mutant enzyme, and negligible for the truncated enzyme form.

Synthesis and characterization of selenotrisulfide-derivatives of lipoic acid and lipoamide.

Thiol-containing compounds, such as glutathione and cysteine, react with selenite under specific conditions to form selenotrisulfides. Previous studies have focused on isolation and characterization of intermolecular selenotrisulfides. This study describes the preparation and characterization of intramolecular selenotrisulfide derivatives of lipoic acid and lipoamide. These derivatives, after separation from other reaction products by reverse-phase HPLC, exhibit an absorbance maximum at 288 nm with an extinction coefficient of 1,500 M(-1) small middle dotcm(-1). The selenotrisulfide derivative of lipoic acid was significantly stable at or below pH 8.0 in contrast to several other previously studied selenotrisulfides. Mass spectral analysis of the lipoic acid and lipoamide derivatives confirmed both the expected molecular weights and also the presence of a single atom of selenium as revealed by its isotopic distribution. The selenotrisulfide derivative of lipoic acid was found to serve as an effective substrate for recombinant human thioredoxin reductase as well as native rat thioredoxin reductase in the presence of NADPH. Likewise, the lipoamide derivative was efficiently reduced by NADH-dependent bovine lipoamide dehydrogenase. The significant in vitro stability of these intramolecular selenotrisulfide derivatives of lipoic acid can serve as an important asset in the study of such selenium adducts as model selenium donor compounds for selenophosphate biosynthesis and as rate enhancement effectors in various redox reactions.

Escherichia coli NifS-like proteins provide selenium in the pathway for the biosynthesis of selenophosphate.

Selenophosphate synthetase (SPS), the selD gene product from Escherichia coli, catalyzes the biosynthesis of monoselenophosphate, AMP, and orthophosphate in a 1:1:1 ratio from selenide and ATP. Kinetic characterization revealed the K(m) value for selenide approached levels that are toxic to the cell. Our previous demonstration that a Se(0)-generating system consisting of l-selenocysteine and the Azotobacter vinelandii NifS protein can replace selenide for selenophosphate biosynthesis in vitro suggested a mechanism whereby cells can overcome selenide toxicity. Recently, three E. coli NifS-like proteins, CsdB, CSD, and IscS, have been overexpressed and characterized. All three enzymes act on selenocysteine and cysteine to produce Se(0) and S(0), respectively. In the present study, we demonstrate the ability of each E. coli NifS-like protein to function as a selenium delivery protein for the in vitro biosynthesis of selenophosphate by E. coli wild-type SPS. Significantly, the SPS (C17S) mutant, which is inactive in the standard in vitro assay with selenide as substrate, was found to exhibit detectable activity in the presence of CsdB, CSD, or IscS and l-selenocysteine. Taken together the ability of the NifS-like proteins to generate a selenium substrate for SPS and the activation of the SPS (C17S) mutant suggest a selenium delivery function for the proteins in vivo.

Selenium-dependent metabolism of purines: A selenium-dependent purine hydroxylase and xanthine dehydrogenase were purified from Clostridium purinolyticum and characterized.

During purification of the selenium-dependent xanthine dehydrogenase (XDH) from Clostridium purinolyticum, another hydroxylase was uncovered that also contained selenium and exhibited similar spectral properties. This enzyme was purified to homogeneity. It uses purine, 2OH-purine, and hypoxanthine as substrates, and based on its substrate specificity, this selenoenzyme is termed purine hydroxylase (PH). The product of hydroxylation of purine by PH is xanthine. A concomitant release of selenium from the enzyme and loss of catalytic activity on treatment with cyanide indicates that selenium is essential for PH activity. Selenium-dependent XDH, also purified from C. purinolyticum, was found to be insensitive to oxygen during purification and to use both potassium ferricyanide and 2,6-dichloroindophenol as electron acceptors. Selenium is required for the xanthine-dependent reduction of 2, 6-dichloroindophenol by XDH. Kinetic analyses of both enzymes revealed that xanthine is the preferred substrate for XDH and purine and hypoxanthine are preferred by PH. This characterization of these selenium-requiring hydroxylases involved in the interconversion of purines describes an extension of the pathway for purine fermentation in the purinolytic clostridia.

Mammalian thioredoxin reductase: oxidation of the C-terminal cysteine/selenocysteine active site forms a thioselenide, and replacement of selenium with sulfur markedly reduces catalytic activity.

Mammalian cytosolic thioredoxin reductase (TrxR) has a redox center, consisting of Cys(59)/Cys(64) adjacent to the flavin ring of FAD and another center consisting of Cys(497)/selenocysteine (SeCys)(498) near the C terminus. We now show that the C-terminal Cys(497)-SH/SeCys(498)-Se(-) of NADPH-reduced enzyme, after anaerobic dialysis, was converted to a thioselenide on incubation with excess oxidized Trx (TrxS(2)) or H(2)O(2). The Cys(59)-SH/Cys(64)-SH pair also was oxidized to a disulfide. At lower concentrations of TrxS(2), the Cys(59)-SH/Cys(64)-SH center was still converted to a disulfide, presumably by reduction of the thioselenide to Cys(497)-SH/SeCys(498)-Se(-). Specific alkylation of SeCys(498) completely blocked the TrxS(2)-induced oxidation of Cys(59)-SH/Cys(64)-SH, and the alkylated enzyme had negligible NADPH-disulfide oxidoreductase activity. The effect of replacing SeCys(498) with Cys was determined by using a mutant form of human placental TrxR1 expressed in Escherichia coli. The NADPH-disulfide oxidoreductase activity of the purified Cys(497)/Cys(498) mutant enzyme was 6% or 11% of that of wild-type rat liver TrxR1 with 5, 5'-dithiobis(2-nitrobenzoic acid) or TrxS(2), respectively, as substrate. Disulfide formation induced by excess TrxS(2) in the mutant form was 12% of that of the wild type. Thus, SeCys has a critical redox function during the catalytic cycle, which is performed poorly by Cys.

Human selenium-dependent thioredoxin reductase from HeLa cells: properties of forms with differing heparin affinities.

The TrxRl form of thioredoxin reductase (TrxR) was the major form of the enzyme isolated from HeLa cells grown in a fermentor at 35 degrees C under controlled aeration conditions favorable to growth, nominally 30% of saturation of dissolved oxygen or 8 ml of oxygen in a liter of medium. This TrxR1 form was not retained on a heparin affinity matrix, it contained one selenium per subunit, was highly active catalytically, and showed strong cross-reactivity with anti-rat liver TrxR1 polyclonal antibodies. At higher aeration, 50% of saturation of dissolved oxygen or 12 ml of oxygen in a liter of medium, HeLa cell growth was slower and additional TrxR forms that bound to heparin were present in purified enzyme preparations. A minor component, TrxR2, the mitochondrial form of TrxR, was detected in the heparin-bound enzyme fraction. One enzyme form that contained less selenium (ca. 0.5 Se per TrxR subunit) was only about 50% as active with thioredoxin or 5,5'dithiobis(2-nitrobenzoic acid) as substrate. Cross-reactivity of this form with anti-rat liver TrxR1 polyclonal antibodies was very weak. The isoelectric point of the low Se enzyme, 5.85, was higher than that, 5.2-5.4, of normal Se content enzyme. Affinity of purified fully active TrxR1 to heparin could be induced by reduction with NADPH or tris-(2-carboxyethyl)phosphine (TCEP). Under anaerobic conditions there was complete retention of Se indicating that an enzyme conformation change effected by reduction was involved. The TCEP-reduced enzyme form was very oxygen labile and upon exposure to air both the Se content and catalytic activity decreased by about 50%. Addition of millimolar concentrations of NADPH or NADP(+) to the TCEP-reduced enzyme gave full protection from oxygen inactivation. TrxR1 exhibited weak peroxidase activity with H(2)O(2) as substrate in the presence of an NADPH-generating system but this activity was unstable. Specific alkylation of the selenocysteine residue of TrxR1 which completely inhibits the NADPH-dependent reduction of disulfides also destroyed peroxidase activity.

Responsiveness of selenoproteins to dietary selenium.

Selenocysteine-containing enzymes that have been identified in mammals include the glutathione peroxidase family (GPX1, GPX2, GPX3, and GPX4), one or more iodothyronine deiodinases and two thioredixin reductases. Selenoprotein P, a glycoprotein that contains 10 selenocysteine residues per 43 kDa polypeptide and selenoprotein W, a 10 kDa muscle protein, are unidentified as to function. Levels of all of these selenocysteine-containing proteins in various tissues are affected to different extents by selenium availability. Increased amounts of selenoproteins observed in response to selenium supplementation were shown in several studies to correlate with increases in the corresponding mRNA levels. In general, selenoprotein levels in brain are less sensitive to dietary selenium fluctuation than the corresponding selenoprotein levels in other tissues.

Levels of major selenoproteins in T cells decrease during HIV infection and low molecular mass selenium compounds increase.

It has been observed previously that plasma selenium and glutathione levels are subnormal in HIV-infected individuals, and plasma glutathione peroxidase activity is decreased. Under these conditions the survival rate of AIDS patients is reduced significantly. In the present study, using 75Se-labeled human Jurkat T cells, we show that the levels of four 75Se-containing proteins are lower in HIV-infected cell populations than in uninfected cells. These major selenoproteins migrated as 57-, 26-, 21-, and 15-kDa species on SDS/PAGE gels. In our earlier studies, the 57-kDa protein was purified from T cells and identified as a subunit of thioredoxin reductase. The 26- and 21-kDa proteins were identified in immunoblot assays as the glutathione peroxidase (cGPX or GPX1) subunit and phospholipid hydroperoxide glutathione peroxidase (PHGPX or GPX4), respectively. We recently purified the 15-kDa protein and characterized it as a selenoprotein of unknown function. In contrast to selenoproteins, low molecular mass [75Se]compounds accumulated during HIV infection and migrated as a diffuse band near the front of SDS/PAGE gels.

Catalytic properties of selenophosphate synthetases: comparison of the selenocysteine-containing enzyme from Haemophilus influenzae with the corresponding cysteine-containing enzyme from Escherichia coli.

The selD gene from Haemophilus influenzae has been overexpressed in Escherichia coli. The expressed protein was purified to homogeneity in a four-step procedure and then carboxymethylated by reaction with chloroacetate. N-terminal sequencing by Edman degradation identified residue 16 as carboxymethyl selenocysteine, which corresponded to the essential cysteine residue in the glycine-rich sequence of the E. coli selenophosphate synthetase. It would be expected that an ionized selenol of a selenocysteine in place of a catalytically essential cysteine residue would result in an enzyme with increased catalytic activity. To test this hypothesis we kinetically characterized the selenocysteine containing selenophosphate synthetase from H. influenzae and compared its catalytic activity to that of the cysteine containing selenophosphate synthetase from E. coli. Our characterization revealed the Km values for the two substrates, selenide and ATP, were similar for both enzymes. However, the selenocysteine-containing enzyme did not exhibit the expected higher catalytic activity. Based on these results we suggest a role of selenocysteine in H. influenzae that is not catalytic.

The NIFS protein can function as a selenide delivery protein in the biosynthesis of selenophosphate.

The NIFS protein from Azobacter vinelandii is a pyridoxal phosphate-containing homodimer that catalyzes the formation of equimolar amounts of elemental sulfur and L-alanine from the substrate L-cysteine (Zheng, L., White, R. H., Cash, V. L., Jack, R. F., and Dean, D. R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2754-2758). A sulfur transfer role of NIFS in which the enzyme donates sulfur for iron sulfur center formation in nitrogenase was suggested. The fact that NIFS also can catalyze the decomposition of L-selenocysteine to elemental selenium and L-alanine suggested the possibility that this enzyme might serve as a selenide delivery protein for the in vitro biosynthesis of selenophosphate. In agreement with this hypothesis, we have shown that replacement of selenide with NIFS and L-selenocysteine in the in vitro selenophosphate synthetase assay results in an increased rate of formation of selenophosphate. These results thus support the view that a selenocysteine-specific enzyme similar to NIFS may be involved as an in vivo selenide delivery protein for selenophosphate biosynthesis. A kinetic characterization of the two NIFS catalyzed reactions carried out in the present study indicates that the enzyme favors L-cysteine as a substrate compared with its selenium analog. A specific activity for L-cysteine of 142 nmol/min/mg compared with 55 nmol/min/mg for L-selenocysteine was determined. This level of enzyme activity on the selenoamino acid substrate is adequate to deliver selenium to selenophosphate synthetase in the in vitro assay system described.

Human thioredoxin reductase from HeLa cells: selective alkylation of selenocysteine in the protein inhibits enzyme activity and reduction with NADPH influences affinity to heparin.

Human thioredoxin reductase (TR) contains selenocysteine (Secys) in a redox center [cysteine (Cys)-497,Secys-498] near the C-terminus. The essential role of Secys in TR isolated from HeLa cells was demonstrated by the alkylation studies. Reaction of native NADPH reduced enzyme with bromoacetate at pH 6.5 inhibited enzyme activity 99%. Of the incorporated carboxymethyl (CM) group, 1.1 per subunit, >90% was in CM-Secys-498. Alkylation at pH 8 increased the stoichiometry to 1.6 per subunit with additional modification of the Cys-59, Cys-64 disulfide center. A minor tryptic peptide containing both CM-Cys-497 and CM-Secys-498 was isolated from enzyme alkylated at pH 6.5 or at pH 8. Preparations of TR isolated from HeLa cells grown in a fermentor under high aeration contained selenium-deficient enzyme species that had 50% lower activity. Decreasing oxygen to an optimal level increased cell yield, and fully active TR containing one Se per subunit was present. Reduction of fully active enzyme with tris-(2-carboxyethyl) phosphine converted it from a low to a high heparin affinity form. The tris-(2-carboxyethyl) phosphine-reduced enzyme was oxygen-sensitive and lost selenium and catalytic activity unless maintained under strictly anaerobic conditions. This enzyme could be converted to an oxygen-insensitive species by addition of NADPH, indicating that bound pyridine nucleotide is important for enzyme stability. An induced enzyme conformation in which the essential Secys is shielded from oxidative damage could explain these effects.

Isotope exchange studies on the Escherichia coli selenophosphate synthetase mechanism.

Selenophosphate synthetase, the Escherichia coli selD gene product, is a 37-kDa protein that catalyzes the synthesis of selenophosphate from ATP and selenide. In the absence of selenide, ATP is converted quantitatively to AMP and two orthophosphates in a very slow partial reaction. A monophosphorylated enzyme derivative containing the gamma-phosphoryl group of ATP has been implicated as an intermediate from the results of positional isotope exchange studies. Conservation of the phosphate bond energy in the final selenophosphate product is indicated by its ability to phosphorylate alcohols and amines to form O-phosphoryl- and N-phosphoryl-derivatives. To further probe the mechanism of action of selenophosphate synthetase, isotope exchange studies with [8-14C]ADP or [8-14C]AMP and unlabeled ATP were carried out, and 31P NMR analysis of reaction mixtures enriched in H218O was performed. A slow enzyme-catalyzed exchange of ADP with ATP observed in the absence of selenide implies the existence of a phosphorylated enzyme and further supports an intermediary role of ADP in the reaction. Under these conditions ADP is slowly converted to AMP. Incorporation of 18O from H218O exclusively into orthophosphate in the overall selenide-dependent reaction indicates that the beta-phosphoryl group of the enzyme-bound ADP is attacked by water with liberation of orthophosphate and formation of AMP. Based on these results and the failure of the enzyme to catalyze an exchange of labeled AMP with ATP, the existence of a pyrophosphorylated enzyme intermediate that was postulated earlier can be excluded.

Selenium-containing formate dehydrogenase H from Escherichia coli: a molybdopterin enzyme that catalyzes formate oxidation without oxygen transfer.

Formate dehydrogenase H, FDH(Se), from Escherichia coli contains a molybdopterin guanine dinucleotide cofactor and a selenocysteine residue in the polypeptide. Oxidation of 13C-labeled formate in 18O-enriched water catalyzed by FDH(Se) produces 13CO2 gas that contains no 18O-label, establishing that the enzyme is not a member of the large class of Mo-pterin-containing oxotransferases which incorporate oxygen from water into product. An unusual Mo center of the active site is coordinated in the reduced Mo(IV) state in a square pyramidal geometry to the four equatorial dithiolene sulfur atoms from a pair of pterin cofactors and a Se atom of the selenocysteine-140 residue [Boyington, J. C., Gladyshev, V. N., Khangulov, S. V., Stadtman, T. C., and Sun, P. D. (1997) Science 275, 1305-1308]. EPR spectroscopy of the Mo(V) state indicates a square pyramidal geometry analogous to that of the Mo(IV) center. The strongest ligand field component is likely the single axial Se atom producing a ground orbital configuration Mo(dxy). The Mo-Se bond was estimated to be covalent to the extent of 17-27% of the unpaired electron spin density residing in the valence 4s and 4p selenium orbitals, based on comparison of the scalar and dipolar hyperfine components to atomic 77Se. Two electron oxidation of formate by the Mo(VI) state converts Mo to the reduced Mo(IV) state with the formate proton, Hf+, transferring to a nearby base Y-. Transfer of one electron to the Fe4S4 center converts Mo(IV) to the EPR detectable Mo(V) state. The Y- is located within magnetic contact to the [Mo-Se] center, as shown by its strong dipolar 1Hf hyperfine couplings. Photolysis of the formate-induced Mo(V) state abolishes the 1Hf hyperfine splitting from YHf, suggesting photoisomerizaton of this group or phototransfer of the proton to a more distant proton acceptor group A-. The minor effect of photolysis on the 77Se-hyperfine interaction with [77Se] selenocysteine suggests that the Y- group is not the Se atom, but instead might be the imidazole ring of the His141 residue which is located in the putative substrate-binding pocket close to the [Mo-Se] center. We propose that the transfer of Hf+ from formate to the active site base Y- is thermodynamically coupled to two-electron oxidation of the formate molecule, thereby facilitating formation of CO2. Under normal physiological conditions, when electron flow is not limited by the terminal acceptor of electrons, the energy released upon oxidation of Mo(IV) centers by the Fe4S4 is used for deprotonation of YHf and transfer of Hf+ against the thermodynamic potential.

Inhibition of NF-kappaB DNA binding and nitric oxide induction in human T cells and lung adenocarcinoma cells by selenite treatment.

NF-kappaB is a major transcription factor consisting of 50(p50)- and 65(p65)-kDa proteins that controls the expression of various genes, among which are those encoding cytokines, cell adhesion molecules, and inducible NO synthase (iNOS). After initial activation of NF-kappaB, which involves release and proteolysis of a bound inhibitor, essential cysteine residues are maintained in the active reduced state through the action of thioredoxin and thioredoxin reductase. In the present study, activation of NF-kappaB in human T cells and lung adenocarcinoma cells was induced by recombinant human tumor necrosis factor alpha or bacterial lipopolysaccharide. After lipopolysaccharide activation, nuclear extracts were treated with increasing concentrations of selenite, and the effects on DNA-binding activity of NF-kappaB were examined. Binding of NF-kappaB to nuclear responsive elements was decreased progressively by increasing selenite levels and, at 7 microM selenite, DNA-binding activity was completely inhibited. Selenite inhibition was reversed by addition of a dithiol, DTT. Proportional inhibition of iNOS activity as measured by decreased NO products in the medium (NO2- and NO3-) resulted from selenite addition to cell suspensions. This loss of iNOS activity was due to decreased synthesis of NO synthase protein. Selenium at low essential levels (nM) is required for synthesis of redox active selenoenzymes such as glutathione peroxidases and thioredoxin reductase, but in higher toxic levels (>5-10 microM) selenite can react with essential thiol groups on enzymes to form RS-Se-SR adducts with resultant inhibition of enzyme activity. Inhibition of NF-kappaB activity by selenite is presumed to be the result of adduct formation with the essential thiols of this transcription factor.