Germinating barley selenium-containing peroxidase is one of the peroxidase isoenzymes

A selenium-containing peroxidase from the germinating barley grown on a selenium-containing artificial medium was isolated and purified by means of cold acetone precipitation, Sephadex-G150 filtration, followed by DEAE-Sepharose chromatography and sodium dodecyl sulfate — polyacrylamid gel electrophoresis. The form of selenium existing in the peptide assayed with paper chromatography was selenomethionine. The amino acid composition of this enzyme was similar to those peroxidases from other sources except amino acids Glu, Val Phe, Lys, and Arg. Electron-spin resonance (ESR) spectra recorded at −136°C showed that both the selenium-containing peroxidase from germinating barley and horseradish peroxidase had same the ESR signals as iron protoporphyine. Those results suggested that the germinating barley selenium-containing peroxidase is one of the peroxidase isoenzymes.     

Uptake,distribution, and turnover rates of selenium in barley

The present communication elucidates initially the topographic distribution of selenium in barley grains. Then by the fluorimetric method the uptake of selenium (selenite) in 8–16 d old germinating barley was estimated. Finally by means of⁷⁵Se the anabolic and catabolic rates (turnover) of⁷⁵Se (selenite) was compared. The distribution of selenium in barley was evaluated after microdissection of barley grains. In dried grains the highest concentration was found in husk and pericarp with about 0.6 ppm Se. Then followed Scutellum with 0.4 and 0.3 ppm in embryon. The aleurone layer, embryonic leaves, and initial root did only have 0.2 ppm Se. In order to know more about the uptake and distribution of selenium in 8-d-old barley, the plants were cultivated for a further 8 d in the culture medium with variation in selenite concentration. In roots and leaves, the uptake did not arrive at saturation during the period studied since the dose-response curve increased up to 0.34 mM selenite in the medium, whereas the selenium levels were about 200 ppm in roots and 30 ppm in leaves. However, the uptake was linear, with concentration during 8 d of cultivation up to 0.84 μM selenite for grain and stem. At higher concentrations the dose-response curve diminished its slope. At 0.34 mM selenite the concentration in grain increased to 6.87 ppm and in the stem to 8.13 ppm. The uptake, distribution, and catabolic rate of selenium components in germinating barley were further evaluated by exposing the plants to 0.0492 μCi⁷⁵Se (12.6 μM selenite) for up to 4 d. Then the plants were moved to a selenium deficient medium for further 4 d. Then finally the medium was supplemented with high doses of cold selenite (0.126 mM selenite) for further 4 d. The first third period made it possible to estimate the rate of uptake. It was highest in roots (313 fmol/h/mg dw), i.e., about 10 times those of grains, stems, and leaves. The intermediate period where the barley was transferred to a selenium deficient medium made it possible to estimate the kinetics and eventual sparing mechanisms. The selenium losses were highest for leaves (39%), then followed by roots and stems (22 and 25%, respectively). The losses were lowest in grain with 9% Se losses. The losses were three times more pronounced during the first day than in the following 3 d. These data may argue that the selenium is distributed into different pools and that sparing mechanisms may be in function. The last period, i.e., the chase experiment, revealed the rate of elimination of selenium under conditions with surplus selenium. The catabolic rate was about 10 times faster in roots (169 fmol/h/mg dw) than in grains and about 8 times faster than in leaves.     

Number and proportion of selenonucleosides in the transfer RNA of Escherichia coli

tRNA isolated from $\text{E}$. $\text{coli}$ grown in a medium containing [⁷⁵Se] sodium selenosulfate was converted to nucleosides and analysed for selenonucleosides on a phosphocellulose column. Upon chromatography of the nucleosides on phosphocellulose column, the radioactivity resolved into three peaks. The first peak consisted of free selenium and traces of undigested nucleotides. The second peak was identified as 4-selenouridine by co-chromatographing with an authentic sample of 4-selenouridine. The identity of the third peak was not established. The second and third peaks represented 93% and 7% of the selenium present in nucleosides respectively.     

Sulfur Compounds in RNA Fraction from Sporulating Cells of Bacillus subtilis

As we reported previously, in the sporulating cells of Bacillus subtilis about 20% of intracellular sulfur is found in the nucleic acid fraction. In the present work further characterization of sulfur compounds in this fraction was made using tracer technique and MAK column chromatography, and changes in pattern of the sulfur compounds during sporulation was observed.

It was found that the greater part of sulfur in the nucleic acid fraction was present as methionine and cysteine, which were associated with tRNA throughout the growth and sporulation. The amount of methionine as methionine tRNA was larger than that of cysteine as cysteine tRNA in the vegetative cells and vice versa in the sporulating cells.     

Isolation and chromatographic behaviour of phenylalanine tRNA from barley embryos

Two fractions of phenylalanine tRNA (tRNA^Phe₁ and tRNA^Phe₂) were purified by BD-cellulose and RPC-5 chromatography of crude tRNA isolated from barley embryos. Successive RPC-5 rechromatography runs of tRNA^Phe₂ showed its conversion into more stable tRNA^Phe₁, suggesting that the two fractions have essentially the same primary structure. Both tRNA^Phe₁ and tRNA^Phe₂ had about the same acceptor activity, but tRNA^Phe₂ was aminoacylated much faster than tRNA^Phe₁. RPC-5 chromatography of crude aminoacylated tRNA showed higher contents of phe-tRNA^Phe₂ than of phe-tRNA^Phe₁ but the ratio of these two fractions estimated by relative fluorescence intensity was about 1. Fluorescence spectra of tRNA^Phe from barley embryos suggest that it contains Y base similar to Y_w from wheat tRNA^Phe.     

Biosynthesis of 5-methylaminomethyl-2-selenouridine, a naturally occurring nucleoside in Escherichia coli tRNA

A selenium-containing nucleoside, 5-methylaminomethyl-2-selenouridine (mnm5se2U), is present in lysine- and glutamate-isoaccepting tRNA species of Escherichia coli. The synthesis of mnm5se2U is optimum (4 mol/100 mol tRNA) when selenium is present at about 1 microM concentration and is neither decreased by a high (8 mM) level of sulfur in the medium nor increased by excessive (10 or 100 microM) levels of selenium. Lysine- and glutamate-isoaccepting tRNA species that contain 5-methylaminomethyl-2-thiouridine (mnm5s2U) coexist with the seleno-tRNAs in E. coli cells and a reciprocal relationship between the mnm5se2U- and the mnm5s2U-containing species is maintained under a variety of growth conditions. The complete 5-methylaminomethyl side chain is not a prerequisite for introduction of selenium at the 2-position. In E. coli mutants deficient in the ability to synthesize the 5-methylaminomethyl substituent, both the 2-thiouridine and the corresponding 2-selenouridine derivatives of intermediate forms are accumulated. Broken cell preparations of E. coli synthesize mnm5se2U in tRNAs by an ATP-dependent process that appears to involve the replacement of sulfur in mnm5s2U with selenium.     

Specific incorporation of selenium into lysine- and glutamate- accepting tRNAs from Escherichia coli

Amino acid transfer nucleic acids (tRNAs) that contain selenium-modified bases are synthesized by Escherichia coli in the presence of low levels (0.1-0.5 microM) of [75Se]selenite or [75Se]selenate. The amount of selenium incorporated (1-2 g atoms/100 mol of tRNA) was unchanged by 10-20-fold variations in selenium or sulfate concentrations or by the addition of 1 mM cysteine, sulfide, or sulfite. Specific incorporation of selenium (as opposed to nonspecific substitution for sulfur) was further indicated by the different reversed phase chromatographic elution patterns of 35S- and 75Se-labeled tRNAs isolated from cells labeled with 35SO2-4 and 75SeO2-4. Also, E. coli mutants unable to synthesize an abundant sulfur-modified base, 4-thiouracil, nevertheless produced normal levels of selenium-modified tRNAs. Two different methods of distinguishing between aminoacylated and nonaminoacylated tRNA, one which examined mobility during reversed phase chromatography and another which employed anti-AMP antibodies, indicated that over 50% of the selenium-containing tRNA had lysine or glutamate acceptor activity.     

The nucleotide sequences of barley cytoplasmic glutamate transfer RNAs and structural features essential for formation of δ-aminolevulinic acid

In chloroplasts and a number of prokaryotes, -aminolevulinic acid (ALA), the universal precursor of porphyrins, is synthesized by a multistep enzymatic pathway with glutamyl-tRNA^Glu as an intermediate. The ALA synthesizing system from barley chloroplasts is highly specific in its tRNA requirement for chloroplast tRNA^Glu; a number of other Glu-tRNAs are inactive in ALA formation although they can be glutamylated by chloroplast aminoacyl-tRNA synthetases. In order to obtain more information about the structural features defining the ability of a tRNA to be recognized by the ALA synthesizing enzymes, we purified and sequenced two cytoplasmic tRNA^Glu species from barley embryos which are inactive in ALA synthesis. By using glutamylated tRNAs as a substrate for the overall reaction, we showed that Glu-tRNA reductase is the enzyme responsible for tRNA discrimination.     

The Effect of Selenite Ions on Growth and Selenium Accumulation in Spirulina platensis

Selenium accumulation and the growth of cyanobacterium Spirulina platensis (Nordst.) Geitl. were studied in a culture with sodium selenite-supplemented nutritional medium. Selenite concentrations below 20 mg/l did not inhibit the growth of S. platensis. The addition of 30 mg/l of this salt somewhat decreased the growth rate during the linear growth phase, induced the earlier suspension transition to the steady-state phase, and substantially lowered the highest optical density of the suspension. However, even at 170 mg/l Na₂SeO₃, the culture still demonstrated a capacity for growth. The content of selenium in the cells depended directly on its concentration in the medium, up to the lethal level. At high selenium concentrations (100–170 mg/l), S. platensis reduced Se(IV) up to Se(0). The latter was secreted onto the cell surface and into the cultural medium. The high concentrations of Na₂SeO₃ acidified the cytoplasmic pH as was measured by ³¹P-NMR spectroscopy. At the same time, the content of protein on a dry weight basis decreased and that of carbohydrates and lipids somewhat increased, just as was observed in S. platensis cells under other stress factors. In the presence of 20 mg/l Na₂SeO₃, the selenium content in the biomass increased by 20000 times as compared to that in the control cells, whereas the biochemical composition of biomass did not change. In this case, the selenium was incorporated almost completely in the protein fraction. The selenium concentration in this fraction increased more significantly when the sulfur content was lowered in the medium.     

Gibberellin estimation and biosynthesis in germinating Hordeum distichon

Gibberellic acid (GA₃) and 13-deoxy-gibberellic acid (GA₇) were identified in extracts of germinating barley as their ¹⁴C-methyl esters. The maximal level of GA₃ was estimated by an isotopic dilution procedure to be 1·5 ng per grain. Germinating barley incorporated 2-¹⁴C-mevalonic acid into several terpenes, whose specific radioactivities were measured, but incorporation into GA₃ could not be detected. Cell-free embryo extracts from germinating barley converted 2-¹⁴C-mevalonic acid into isopentenol, dimethylallyl alcohol, farnesol and squalene, while ¹⁴C-isopentenyl pyrophosphate was incorporated into geraniol, farnesol, geranylgeraniol and squalene. There was no detectable incorporation into the gibberellin intermediate ent-kaurene.     

Isolation and characterization of a selenium metabolism mutant of Salmonella typhimurium.

Selenium is a constituent in Escherichia coli of the anaerobic enzyme formate dehydrogenase in the form of selenocysteine. Selenium is also present in the tRNA of E. coli in the modified base 5-methylaminomethyl-2-selenouracil (mnm5Se2U). The pathways of bacterial selenium metabolism are largely uncharacterized, and it is unclear whether nonspecific reactions in the sulfur metabolic pathways may be involved. We demonstrated that sulfur metabolic pathway mutants retain a wild-type pattern of selenium incorporation, indicating that selenite (SeO32-) is metabolized entirely via selenium-specific pathways. To investigate the function of mnm5Se2U, we isolated a mutant which is unable to incorporate selenium into tRNA. This strain was obtained by isolating mutants lacking formate dehydrogenase activity and then screening for the inability to metabolize selenium. This phenotype is the result of a recessive mutation which appears to map in the general region of 21 min on the Salmonella typhimurium chromosome. A mutation in this gene, selA, thus has a pleiotropic effect of eliminating selenium incorporation into both protein and tRNA. The selA mutant appears to be blocked in a step of selenium metabolism after reduction, such as in the actual selenium insertion process. We showed that the absence of selenium incorporation into suppressor tRNA reduces the efficiency of suppression of nonsense codons in certain contexts and when wobble base pairing is required. Thus, one function of mnm5Se2U in tRNA may be in codon-anticodon interactions.     

Lysyl tRNAs of lupinus luteus seeds

Lysine accepting transfer RNA of lupin seeds and lupin embryo axes can be fractionated into at least 5 species by reversed-phase chromatography (RPC-5). One main and two minor isoacceptors were observed in wheat and barley embryos. Changes in isoaccepting species of tRNA^1ys were followed in cotyledons of germinating lupin seedlings. Ribosome binding studies revealed that one of the main lupin tRNA^1ys species recognizes the AAG codon, the second AAA and the third one AAA and AAG.     

The Methylated Purines of Tetrahymena pyriformis Transfer Ribonucleic Acid*

SYNOPSIS. By phenol extraction and DEAE cellulose column chromatography, tRNA was isolated from Tetrahymena pyriformis strain GL. Following acid hydrolysis of the tRNA, the methylated purine content was determined by Dowex 50 column chromatography and paper chromatography. The most abundant methylated guanine derivative was found to be N²-DMG. Also present were 1-MG, N²-MG, and 7-MG. The most abundant methylated adenine was found to be 1-MA; no 2-MA was detected. Small amounts of the N⁶-methyladenines were detected.     

Selenium-containing peroxidases of germinating barley

Germinating barley grown on an artificial medium was exposed to⁷⁵Se-selenite for 8 d. Then the leaves were homogenized and proteins were separated by means of Sephadex G-150 filtration, followed by DEAE-Sepharose chromatography. Each fraction collected was assayed for total protein, radioactivity, and peroxidase activity. In barley leaves, three protein peaks (peaks no. I, II, and III) with peroxidase activity could be separated by Sephadex G 150 filtration. Each fraction was then further separated on DEAE-Sepharose chromatography. Thus, peaks I and II were resolved by DEAE-Sepharose into one major and two minor peaks of radioactivity. However, only the major peak showed peroxidase activity. Peak III was resolved from the gel filtration on the DEAE-sepharose into one major and four minor peaks of radioactivity. The major and three of the minor radioactivity peaks contained peroxidase activity. The protein fractions were separated by polyacrylamide gel electrophoresis. The molecular weights of separated proteins were estimated by means of molecular markers, and⁷⁵Se radioactivity was evaluated by autoradiography. Thus, gel filtration peak I contained four bands with mol wts of 128, 116, 100, and 89 kDa. Of these, the 89 kDa protein contained selenium. Peak II contained three protein bands, with mol wts 79.4, 59.6, and 59.9. The 59.6 band was a selenoprotein. Peak III contained four protein bands (and some very weak bands). The four major bands had mol wts of 38.6, 31.6, 30.2, and 29.2 kDa. The last mentioned band was a selenoprotein.     

Speciation, Distribution, and Bioavailability of Soil Selenium in the Tibetan Plateau Kashin–Beck Disease Area—A Case Study in Songpan County, Sichuan Province, China

To clarify the relationship between the soil selenium distribution and its bioavailability with the distribution of Kashin–Beck disease (KBD) endemic areas on the eastern edge of the Tibetan Plateau, samples of natural soil (0–20 cm), cultivated topsoil, and main crops of the region (highland barley) were collected at different altitudes according to topographical and geomorphological features in both KBD and non-KBD areas of Songpan County. These samples were used for determination and analysis of total selenium content in soil and highland barley and available selenium that can be absorbed and utilized by plants. The results showed that the average total selenium content of natural and cultivated topsoil in KBD areas was lower than that in non-KBD areas (natural soil, P?=?0.061; cultivated soil, P?=?0.002), which is in agreement with the geographical distributions of selenium in other KBD-affected areas. However, the total soil selenium content exhibits certain micro-spatial distribution features, namely, the total selenium content in some endemic areas was significantly higher than that of non-KBD areas. This result was contrary to the general distribution that total selenium content in a KBD-affected area is lower than that in a non-KBD area. We further studied the extraction rate and content of soil selenium in six different fractions. The results indicated that the content and extraction rate of available selenium in KBD-affected areas were significantly lower than those in non-KBD areas. There is a distinct positive correlation between plant-available selenium and highland barley selenium (r?=?0.875, P?=?0.001) and a distinct negative correlation with altitude (r?=??0.801, P?=?0.010). Therefore, in KBD endemic areas, the selenium content in crops decreases as the available selenium content in soil decreases and is closely related to the geographical environment features (such as altitude and precipitation). These results suggest that the soil available selenium and ecological features are important factors that restrict the dietary selenium flux for residents in KBD endemic areas of the Tibetan Plateau, providing a theoretical and experimental basis for implementing agricultural measures to regulate the ecological cycle of the selenium flux in the KBD endemic area.     

Functional diversity of the rhodanese homology domain: the Escherichia coli ybbB gene encodes a selenophosphate-dependent tRNA 2-selenouridine synthase

Escherichia coli has eight genes predicted to encode sulfurtransferases having the active site consensus sequence Cys-Xaa-Xaa-Gly. One of these genes, ybbB, is frequently found within bacterial operons that contain selD, the selenophosphate synthetase gene, suggesting a role in selenium metabolism. We show that ybbB is required in vivo for the specific substitution of selenium for sulfur in 2-thiouridine residues in E. coli tRNA. This modified tRNA nucleoside, 5-methylaminomethyl-2-selenouridine (mnm(5)se(2)U), is located at the wobble position of the anticodons of tRNA(Lys), tRNA(Glu), and tRNA(1)(Gln). Nucleoside analysis of tRNAs from wild-type and ybbB mutant strains revealed that production of mnm(5)se(2)U is lost in the ybbB mutant but that 5-methylaminomethyl-2-thiouridine, the mnm(5)se(2)U precursor, is unaffected by deletion of ybbB. Thus, ybbB is not required for the initial sulfurtransferase reaction but rather encodes a 2-selenouridine synthase that replaces a sulfur atom in 2-thiouridine in tRNA with selenium. Purified 2-selenouridine synthase containing a C-terminal His(6) tag exhibited spectral properties consistent with tRNA bound to the enzyme. In vitro mnm(5)se(2)U synthesis is shown to be dependent on 2-selenouridine synthase, SePO(3), and tRNA. Finally, we demonstrate that the conserved Cys(97) (but not Cys(96)) in the rhodanese sequence motif Cys(96)-Cys(97)-Xaa-Xaa-Gly is required for 2-selenouridine synthase in vivo activity. These data are consistent with the ybbB gene encoding a tRNA 2-selenouridine synthase and identifies a new role for the rhodanese homology domain in enzymes.     

Salvage of the nucleic acid base queuine from queuine-containing TRNA by animal cells

The incorporation of queuine into tRNA and its fate upon tRNA turnover has been studied in the Vero and L-M cell lines. An assay was developed using [³H]dihydroqueuine to detect the queuine acceptance and, thus, the queuine content of tRNA in intact cells. While L-M cells can use only queuine, Vero cells can use either queuine or its nucleoside, queuosine, to form queunine-containing tRNA. Since queuosine is not a substrate for the enzyme which incorporates queuine into tRNA, Vero cells must generate queuine from its nucleoside. When Vero cells are labelled with [³H]dihydroqueuine, the half life of acid insoluble radioactivity is 52 days in queuine-free medium and 3.1 days in queuine-containing medium, indicating that [³H]dihydroqueuine is salvaged from tRNA and reused by Vero cells, but that exogenous queuine can compete with the salvaged [³H]dihydroqueuine. When L-M cells are labelled with [³H]dihydroqueuine, the half life of the acid insoluble radioactivity is 1.2 days in the presence or absence of queuine, indicating the absence of queuine salvage in L-M cells.     

The biosynthesis of chlorophyll in greening barley (Hordeum vulgare). Is there a light-independent protochlorophyllide reductase?

Recently, some evidence for the occurence of a light-independent protochlorophyllide-reducing enzyme in greening barley plants has been presented. In the present work this problem was reinvestigated. -[¹⁴C] Aminolevulinic acid was fed to isolated barley shoots from plants which had been preilluminated for various lengths of time. Porphyrins which had been synthesized during the dark incubation were analyzed by high-performance liquid chromatography. There was no evidence for a light-independent synthesis of chlorophyll(ide). The ¹⁴C-labelled precursor was incorporated almost exclusively into protochlorophyllide. The reduction of labelled protochlorophyllide to chlorophyllide was strictly light-dependent. These results are not consistent with the existence of a light-independent protochlorophyllide-reductase in barley as proposed previously.Abbreviation HPLC high-performance liquid chromatography     

Purification and partial characterization of a glutamyl-tRNA synthetase from the unicellular green algaScenedesmus obliquus,mutant C-2A′

The synthesis of 5-aminolevulinic acid commences with the ligation of glutamate to a specific tRNA^Glu by a glutamyl-tRNA synthetase (E.C. 6.1.1.17) (Huang et al., 1984, Science 225, 1482–1484). The synthetase from the yellow pigment mutant C-2A of the unicellular green alga Scenedesmus obliquus was purified by sequential column chromatography on Sephacryl S-300, Blue Sepharose, phosphocellulose P11 and by fast protein liquid chromatography (FPLC) on Mono Q. After denaturing sodium dodecylsulfate (SDS)-gel electrophoresis the purified enzyme preparation revealed a single protein band with a molecular mass of 55 kDa, proving the apparent homogeneity of the glutamyl-tRNA synthetase. A molecular mass of 105 ± 10 kDa was determined for the native protein by chromatography on Sephadex G-150. From these data it can be concluded that the glutamyl-tRNA synthetase from S. obliquus is a homodimer. The purified protein is active within a pH range from 7.0 to 9.0 with a maximum activity at pH 8.0. Kinetics for the binding of glutamate to the tRNA, performed with highly purified enzyme preparations, showed a K _m value of 2.3 M ± 0.3 for glutamate.Abbreviations ALA 5-aminolevulinic acid - FPLC fast protein liquid chromatography - Glu glutamate - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - SDS sodium dodecylsulfate - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-glycine This work was supported by a grant of the Deutsche Forschungsgemeinschaft. U.C. Vothknecht is grateful for a Nachwuchs-förderungsstipendium des Landes Hessen. The authors want to thank Ms. B. Böhm, J. Gade and K. Eckhardt for skillful technical assistance. The authors also want to thank Dr. C.G. Kannangara (Carlsberg Institute, Kopenhagen, Denmark) for the donation of tRNA from barley and Dr. D. Jahn (FB Biology/Microbiology, Philipps-University, Marburg, FRG) for the tRNA^Glufrom E. coli.