Plasma selenium in specific and non-specific forms

Selenium is present in plasma and tissues in specific and non-specific forms. The experiments reported here were carried out to clarify some factors that affect these forms of the element in plasma. A selenium-replete human subject was given 400 microg of selenium daily for 28 days as selenomethionine and, in a separate experiment, as selenate. The selenomethionine raised plasma and albumin selenium concentrations. Selenate did neither. The molar ratio of methionine to selenium in albumin was approximately 8000 under basal and selenate-supplemented conditions but 2800 after selenomethionine supplementation. This demonstrates that selenium from selenomethionine, but not selenium from selenate, can be incorporated into albumin, presumably as selenomethionine in the methionine pool. Selenocysteine incorporation into albumin was studied in rats using (75)Se-selenocysteine. No evidence was obtained for incorporation of (75)Se into albumin after exogenous administration or endogenous synthesis of (75)Se-selenocysteine. Thus, selenocysteine does not appear to be incorporated non-specifically into proteins as is selenomethionine. These findings are in support of selenomethionine being a non-specific form of selenium that is metabolized as a constituent of the methionine pool and is unaffected by specific selenium metabolic processes. No evidence was found for non-specific incorporation of selenium into plasma proteins when it was administered as selenate or as selenocysteine. These forms of the element appear to be metabolized by specific selenium metabolic processes.     

A comparison of the uptake of [75Se]selenite, [75Se]selenomethionine and [35S]methionine by tissues of ewes and lambs.

The fate of selenium, given as Na2(75)SeO3, or [75Se]selenomethionine, and of [35S]methionine administered intravenously to ewes and lambs, has been examined. The main intention was to follow the incorporation of selenium into protein in a number of tissues, including liver and kidney, and to measure the extent of that incorporation of selenoamino acid, particularly with respect to the administration of selenite. The ewes chosen were lactating ewes with lambs at foot, and the lambs were animals which had been weaned on to fodder low in selenium and were recovering from white muscle disease with selenium therapy. These two experimental situations were chosen as they offered conditions under which selenium incorporation might be considered to be maximal. Entry of isotope into milk was rapid and was greater when 75Se was given as the selenoamino acid than as selenite. In both ewes and lambs greater amounts of activity, derived from selenite, were bound to plasma proteins than to the proteins of milk. This was particularly evident in samples taken some hours after administration. This ability of the plasma to bind selenium was demonstrated by alkaline dialysis. Small, though significant amounts of selenium, derived from Na2(75)SeO3, were incorporated as selenoamino acids into the proteins of liver, kidney and pancreas, as well as into the proteins of milk and plasma. In ewes, both selenomethionine and selenocystine were identified chromatographically in enzyme digests of defatted liver and kidney. Some differences occurred in the distribution of labelled compounds in organs from lactating ewes and recovering lambs. The incorporation of selenium into protein is discussed briefly in relation to the recent findings of an association between selenium and the enzyme glutathione peroxidase.     

In Vitro Incorporation of Selenomethionine into Protein by Vigna radiata Polysomes

Vigna radiata polysomes efficiently incorporated [⁷⁵Se]selenomethionine, [¹⁴C]methionine, and [¹⁴C]leucine in vitro. The optimal conditions for translation were determined to be 4.8 millimolar Mg²⁺, 182 millimolar K⁺, and pH 7.4. The rates of incorporation of [⁷⁵Se]selenomethionine and [¹⁴C]methionine were similar when measured separately, but [⁷⁵Se]selenomethionine incorporation was 35% less than [¹⁴C]methionine incorporation when both amino acids were present in equal molar concentrations. Polyacrylamide gel electrophoresis of the hot trichloroacetic acid precipitable translation products demonstrated synthesis of high molecular weight labeled proteins in the presence of [⁷⁵Se]selenomethionine or [³⁵S]methionine. No major differences in molecular weights could be detected in the electrophoretic profiles. Utilization of selenomethionine during translation by Vigna radiata polysomes establishes a route for the assimilation of selenomethionine by plants susceptible to selenium toxicity.     

Optimization of an Escherichia coli formate dehydrogenase assay for selenium compounds.

A microbiological assay to detect different chemical compounds of selenium for potential future use in the study of the distribution of these chemical forms in foods is being developed. This assay is based on the detection, by infrared analysis, of CO2 in a culture of Escherichia coli when the bacteria are grown in the presence of various selenium compounds. The CO2 production is the result of selenium-dependent formate dehydrogenase activity, which catalyzes oxidation of formic acid produced during glucose metabolism. Smooth response curves were generated over several orders of magnitude for selenocystine, selenite, and selenomethionine. The assay detects selenium concentrations (above background) as low as 1.5 nM for selenocystine and selenite and 4 nM for selenomethionine in minimal medium. Detection of selenomethionine was enhanced (to a sensitivity of 1.5 nM) by the addition of methionine to minimal medium and was enhanced even further (to a sensitivity of 0.8 nM) by the addition of a defined mixture of amino acids. Selenomethionine could be assayed in the presence of an amino acid concentration which is proportional to the amino acid/elemental selenium ratio found in a wheat gluten reference material (NIST SRM 8418). This implies that the assay can detect selenium compounds in a variety of foods at low concentrations, avoiding the background CO2 production caused by high concentrations of non-selenium-containing amino acids. The observation that methionine enhanced selenomethionine availability for formate dehydrogenase synthesis supports studies in animals demonstrating that methionine controls selenomethionine incorporation into selenoenzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Catalytic action of L-methionine gamma-lyase on selenomethionine and selenols.

We examined the catalytic action of L-methionine gamma-lyase (EC 4.4.1.11) on selenomethionine (2-amino-4-(methylseleno)butyric acid), methaneselenol, l-hexaneselenol, and benzeneselenol. The enzyme catalyzes alpha, gamma-elimination of selenomethionine to yield alpha-letobutyrate, ammonia, and methaneselenol, and also its gamma-replacement reaction with various thiols to produce S-substituted homocysteines. Selenomethionine is an even better substrate than methionine in alpha, gamma-elimination but is less effective in gamma-replacement. In addition, L-methionine gamma-lyase catalyzes gamma-replacement reaction of methionine and its derivatives with selenols to form the corresponding Se-substituted selenohomocysteines, although selenols are less efficient substituent donors than thiols. This is the first proven mechanism for the incorporation of selenium atom into amino acids.     

Se-(8-azidoadenosyl)[75Se]selenomethionine as a photoaffinity label for S-adenosylmethionine binding proteins.

A method is described for the synthesis and purification of the photoaffinity label Se-(8-azidoadenosyl)[75Se]selenomethionine. This photoaffinity label can be used to specifically and covalently label the S-adenosylmethionine binding site of proteins that use this cofactor, as exemplified by labeling of thioether methyltransferase. By utilizing the gamma-emitting isotope of selenium, Se-(8-azidoadenosyl)[75Se]selenomethionine eliminates the need for the impregnation of acrylamide gels with fluorographic enhancers and dilution of liquid samples into scintillation cocktails, as is required with the commonly used methyl-3H-labeled and 35S-labeled S-(8-azidoadenosyl)methionine.     

The path of unspecific incorporation of selenium in Escherichia coli

The path of unspecific selenium incorporation into proteins was studied in Escherichia coli mutants blocked in the biosynthesis of cysteine and methionine or altered in its regulation. Selenium incorporation required all enzymatic steps of cysteine biosynthesis except sulfite reduction, indicating that intracellular reduction of selenite occurs nonenzymatically. Cysteine (but not methionine) supplementation prevented unspecific incorporation of selenium by repressing cysteine biosynthesis. On the other hand, when the biosynthesis of cysteine was derepressed in regulatory mutants, selenium was incorporated to high levels. These findings and the fact that methionine auxotrophic strains still displayed unspecific incorporation show that selenium incorporation into proteins in E. coli occurs mainly as selenocysteine. These findings also provide information on the labeling conditions for incorporating ⁷⁵Se only and specifically into selenoproteins. Received: 2 May 1997 / Accepted: 23 June 1997     

The synthesis of β-galactosidase inEscherichia coli in the presence of Methionine analogues

The effect of methionine analogues, selenomethionine and selenoethionine, on the synthesis of β-galactosidase inEscherichia coli was investigated. It was found that the incorporation of selenomethionine into β-galactosidase results in the formation of a protein exhibiting normal enzyme activity and immunologically cross-reacting with the antiserum against normal β-galactosidase. However, the selenomethionine enzyme was found to be more susceptible to heat, urea and trypsin. On the other hand, an immunologically cross-reacting protein without the enzyme activity was synthesized in the presence of selenoethionine. The results obtained seem to support the idea that the presence of methionine methyl groups is essential for the activity of β-galactosidase, whereas methionine sulphur can be replaced without changing the enzyme activity.     

Selenomethionine and selenocysteine double labeling strategy for crystallographic phasing

A protocol for the quantitative incorporation of both selenomethionine and selenocysteine into recombinant proteins overexpressed in Escherichia coli is described. This methodology is based on the use of a suitable cysteine auxotrophic strain and a minimal medium supplemented with selenium-labeled methionine and cysteine. The proteins chosen for these studies are the cathelin-like motif of protegrin-3 and a nucleoside-diphosphate kinase. Analysis of the purified proteins by electrospray mass spectrometry and X-ray crystallography revealed that both cysteine and methionine residues were isomorphously replaced by selenocysteine and selenomethionine. Moreover, selenocysteines allowed the formation of unstrained and stable diselenide bridges in place of the canonical disulfide bonds. In addition, we showed that NDP kinase contains a selenocysteine adduct on Cys122. This novel selenium double-labeling method is proposed as a general approach to increase the efficiency of the MAD technique used for phase determination in protein crystallography.     

Selenoprotein inAspergillus terreus

Aspergillus terreus, a moderately selenium-tolerant fungus, metabolized⁷⁵ Se-selenite into several protein seleno-amino acids: selenomethionine and selenocysteine, as well as, nonprotein seleno-amino acids, selenocystathionine, and y-glutamyl selenomethyl selenocysteine. The results indicate the failure of the fungus to discriminate between sulphur and selenium. Selenium was also incorporated into several proteins of different molecular weights, mostly of low molecular weight proteins. Labeled studies showed the presence of high levels of selenomethionine and selenocysteine in the protein hydrolysate. The actual incorporation of protein selenoamino acids into the fungal protein was proven. The results demonstrated a finding that detracts from previous held views.     

Selenium supplementation of infant formula: uptake and retention of various forms of selenium in suckling rats

Formula-fed infants often have lower serum selenium levels than breast-fed infants. Although no deleterious effects have been correlated to this finding, supplementation of formula with selenium is considered. In this study, we investigated the uptake and retention by suckling rat pups of ⁷⁵Se from selenite, selenate, and selenomethionine added to infant formula. The molecular distribution of ⁷⁵Se in liver, kidney, intestine, and plasma was followed by gel-filtration chromatography on Superose 12. ⁷⁵Se-uptake was most rapid from selenomethionine (70% at 1 hr), followed by selenate (51%) and selenite (29%). This difference was explained by a higher retention of ⁷⁵Se in the stomach and small intestinal wall of pups given selenite supplement. Plasma distribution of ⁷⁵Se as studied by gel filtration was also different, with a higher proportion of ⁷⁵Se from selenomethionine being protein-bound than from selenite or selenate. Similarly, a larger proportion of ⁷⁵Se from selenomethionine became protein-bound in the liver than from selenite or selenate. In conclusion, although whole body retention after 24–48 hr was similar, the metabolic fate of selenium varies considerably with the form of selenium added to formula. Further studies are needed to study the long-term consequences of selenium accumulated in different body compartments.     

Investigation of the form of selenium in the hydrogenase from chemolithotrophically cultured Bradyrhizobium japonicum

It has been established that the hydrogenase from autotrophically cultured Bradyrhizobium japonicum contains selenium as a bound constituent. About 80% of the enzyme selenium remains bound during precipitation with 5% trichloroacetic acid (TCA). However, 85% of the selenium bound to the enzyme is released by a combined treatment of urea, heat and TCA. Neither selenomethionine nor selenocysteine could be detected on analysis of anaerobically hydrolyzed enzyme. These results are consistent with the report showing that the structural genes for this enzyme do not contain a TGA codon (Sayavedra-Soto et al. 1988) which has been reported to code for selenocysteine incorporation into several proteins (Chambers et al. 1986; Zinoni et al. 1986; Stadtman 1987). We have demonstrated that ⁷⁵Se from the labeled hydrolyzed enzyme forms the derivative' selenodicysteine. The form of selenium resulting in the synthesis of this derivative apparently is SeO _inf3 ^sup= or a compound such as Se⁼ which is easily oxidized to SeO _inf3 ^sup= . In a separate approach it was established that 12–16% of the total ⁷⁵Se in the native enzyme reacted with 2,3-diaminonaphthalene indicating that this fraction was present as SeO _inf3 ^sup= . The remaining ⁷⁵Se was bound to the enzyme protein. From this research, we concluded that Se in Bradyrhizobium japonicum hydrogenase is present in a labile bound form. In this respect, this enzyme is similar to xanthine dehydrogenase and nicotinic acid hydroxylase, both of which contain labile Se constituents that have not been defined.Technical paper no. 8980 from the Oregon Agricultural Experiment Station     

Failure of selenomethionine residues in albumin and immunoglobulin G to protect against peroxynitrite.

Selenomethionine has been suggested to protect against peroxynitrite by quenching it in vivo. Selenomethionine is distributed randomly in the methionine pool. Albumin and IgG were purified from plasma of a human being before and after 28 days of supplementation with 400 microg selenium/day as selenomethionine. The albumin contained 1 selenium atom, presumably as selenomethionine, per 8000 methionine residues before supplementation and 1 per 2800 after supplementation. Although this ratio suggested that selenomethionine would not have as great an effect in quenching peroxynitrite as would methionine, direct testing of the albumin and IgG fractions was carried out to assess the ability of these proteins to prevent peroxynitrite oxidation of dihydrorhodamine 123 to rhodamine 123. The ability of the albumin preparations to resist nitration of tyrosine residues was also assessed. The high-selenomethionine preparations of the proteins had no greater effect in quenching the peroxynitrite than did the normal-selenomethionine preparations. These results do not support the proposal that selenomethionine in proteins contributes to in vivo protection against peroxynitrite.     

Incorporation of selenium from selenite and selenocystine into glutathione peroxidase in the isolated perfused rat liver

The erythrocyte-free, isolated perfused rat liver was used to study the incorporation of selenium into glutathione peroxidase. Gel filtration and ion exchange chromatography of liver supernatant demonstrated ⁷⁵Se incorporation into glutathione peroxidase. A 9-fold excess of unlabelled selenium as selenite or selenide very effectively reduced ⁷⁵Se incorporation from L[⁷⁵Se]-selenocystine, but a 100-fold excess of unlabelled selenium as selenocystine was relatively ineffective as compared to selenite or selenide in diluting ⁷⁵Se incorporation from [⁷⁵Se]selenite. These results indicate that selenide and selenite are more readily metabolized than is selenocysteine to the immediate selenium precursor used for glutathione peroxidase synthesis, and suggest a posttranslational modification at another amino acid residue, rather than direct incorporation of selenocysteine, as the mechanism for formation of the presumed selenocysteine moiety of the enzyme.     

Control of isocitrate lyase synthesis in Chlorella fusca var. vacuolata. Rate of enzyme synthesis in the presence and absence of acetate measured by [35S]methionine labelling and immunoprecipitation.

The rate of increase of isocitrate lyase activity was measured in darkened Chlorella fusca var. vaculoata cultures in the presence and absence of acetate and compared with the rate of incorporation of [35S]methionine into isocitrate lyase enzyme protein under the same conditions. Isocitrate lyase enzyme protein was isolated for this purpose by specific immunoprecipitation and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. After 4h in the dark, in the presence of acetate the rate of increase of isocitrate lyase activity was 75 times that in the absence of acetate. Incorporation of [35S]methionine into isocitrate lyase was 140 times greater in the presence of acetate. Incorporation of [35S]methionine into the trichloroacetic acid-insoluble fraction overall was about five times as fast in the presence of acetate. These data are not consistent with an increased turnover of isocitrate lyase enzyme molecules, sufficient to account for the low rate of increase of isocitrate lyase activity in the absence of acetate. The greater rate of enzyme synthesis in the presence of acetate must therefore be due to some effect of this metabolite on the processing or translation of isocitrate lyase mRNA.     

The metabolism of selenite and selenomethionine in mouse fibroblasts grown in tissue culture

Recent reports have provided evidence that selenium is an essential growth factor for cells grown in tissue culture. The aim of the work reported in this paper was to evaluate mouse fibroblasts as a model for the study of selenium metabolism in mammalian cells. The results showed that transformed mouse lung fibroblasts grown in media containing 9.1% bovine serum did not show a growth response to added selenium as selenite over the range of 10–1000 ng/mL. Uptake of selenium by cells was a direct function of the selenium concentration in the medium. The rate of uptake varied with the time of exposure of the cells to the selenium, and to the form of selenium in the medium. Experiments using radioactive selenium showed that⁷⁵Se from selenite was rapidly absorbed into the cell wall, but slowly incorporated into the soluble protein fraction.⁷⁵Se from selenomethionine was more slowly absorbed into the cells, but once inside, it became rapidly incorporated into soluble cytoplasmic proteins. Cell fractionation and gel filtration procedures established that⁷⁵Se from selenite was rapidly incorporated into glutathione peroxidase (GSHpx), whereas⁷⁵Se from selenomethionine was initially incorporated into a wide spectrum of proteins and only after a longer period did the⁷⁵Se peak become associated with GSHpx. These findings suggest fundamental differences exist in the manner in which mammalian cells initially absorb and metabolize different selenium compounds.     

Induction of retrovirus gene expression by selenium compounds

Sodium selenite, sodium selenate, selenium oxide, selenophypoxanthine, selenopurine, selenocysteine, selenoethionine and selenomethionine were tested for their ability to induce endogenous retrovirus expression in cultured AKR mouse embryo fibroblasts. All except selenoethionine were highly toxic to the cells. Only selenomethionine however, had the ability to induce virus expression under the conditions used. The level of virus induction (plaque-forming-units/10(5) cells) was roughly proportional to dose over the range of concentrations from 0.25 mM to 5.0 mM. Induction was best observed when a treatment duration of 48 h was used and required the treatment of actively dividing cells. The induction and the cytotoxic effects of selenomethionine could be abrogated by simultaneous treatment with methionine. A ratio of methionine to selenomethionine of 1:10 inhibited induction by approx. 60% while equivalent amounts of methionine inhibited selenomethionine-mediated induction by greater than 96%, indicating that methionine was more efficiently recognized by the cells than was selenomethionine. A possible mechanism for selenomethionine induction involving the production of undermethylated DNA is presented.     

Overexpression, purification, and characterization of selenomethionyl farnesyl diphosphate synthase of Bacillus stearothermophilus

To facilitate X-ray crystal structure solution of farnesyl diphosphate (FPP) synthase of Bacillus stearothermophilus, selenomethionyl recombinant enzyme was overproduced in a methionine (Met) auxotrophic strain of Escherichia coli, and purified to homogeneity by two chromatographic steps. About 50 mg of the pure selenomethionyl enzyme was obtained from 2 g of E. coli cells. Inductively coupled plasma (ICP) emission spectrometric analysis for selenium content showed that all of the Met residues in the FPP synthase were substituted by selenomethionine (SeMet). The selenomethionyl recombinant enzyme showed similar chromatographic behavior, heat stability, immunochemical property, product specificity, and kinetic parameters to those of the wild-type enzyme, indicating that SeMet substitution has little effect on the prenyltransferase with respect to substrate binding, enzymatic activity, and structure.     

Interactions of Methionine and Selenomethionine with Methionine Adenosyltransferase and Ethylene-generating Systems

Since selenomethionine appears to be a better precursor of ethylene in senescing flower tissue of Ipomoea tricolor and in indole acetic acid-treated pea stem sections than is methionine (Konze JR, N Schilling, H Kende 1978 Plant Physiol 62: 397-401), we compared the effectiveness of selenomethionine and methionine to participate in reactions which may be connected to ethylene biosynthesis. Evidence is presented that selenomethionine is also a better substrate of methionine adenosyltransferase (ATP: methionine S-adenosyltransferase, EC 2.5.1.6) from I. tricolor, the V_max for selenomethionine being twice as high as that for methionine. The affinity of the enzyme is higher for methionine than for selenomethionine, however. Methionine added to flower tissue together with selenomethionine inhibits the enhancement of ethylene synthesis by the seleno analog. Likewise, methionine reduces the high, selenomethionine-dependent reaction rates of methionine adenosyltransferase from I. tricolor flower tissue. On the other hand, selenomethionine is less effective as an ethylene precursor than is methionine in model systems involving oxidation by free radicals. It was concluded that activation of methionine by methionine adenosyltransferase and formation of S-adenosylmethionine are more likely to be involved in ethylene biosynthesis than is oxidation of methionine by free radicals.     

In vitro incorporation of selenomethionine into protein by astragalus polysomes

Selenium-accumulator plants synthesize selenium compounds that differ from those produced by nonaccumulators. To determine if there are any subcellular differences between accumulators and nonaccumulators in the use of selenomethionine in vitro, polysomes from Astragalus crotalariae (accumulator) and Astragalus lentiginosis (nonaccumulator) were translated in the presence of selenomethionine. Polysomes from both species efficiently used selenomethionine in vitro during the translation process. Inasmuch as no differences in the incorporation of selenomethionine into protein were observed between polysomes from the two types of Astragalus, it can be inferred that in accumulators there exists a mechanism that either prevents synthesis of selenomethionine or modifies this selenocompound to a derivative that cannot be incorporated into protein.