Selenium-containing proteins of rat kidney

Rat kidney selenium (Se)-containing proteins were studied by isotopic labeling with [75Se]selenite or [75Se]selenomethionine via three routes: oral, intraperitoneal injection, and incubation of kidney slices with the isotope. The two major Se-containing proteins in kidney were fractionated and partially characterized. 75Se elution profiles from Sephadex G-150 chromatography were similar for each labeling protocol, except for the profile obtained following incubation of slices with [75Se]selenomethionine. Of the two major 75Se-containing proteins, the one eluting at the void volume during Sephadex G-150 fractionation had a subunit of 23,000 Mr. The 75Se-labeled tryptic peptide from this protein and a 75Se-containing tryptic peptide from glutathione peroxidase had the same elution time from an HPLC column. A 75,000 Mr 75Se-containing protein had a 65,000 Mr subunit, and the 75Se-labeled tryptic peptide from this protein eluted from the HPLC column before that of glutathione peroxidase. Glutathione peroxidase is the most abundant kidney selenoprotein. Injection of animals with 75Se is the method of choice for isotopic labeling of rat kidney Se-containing proteins. Appropriate methods were developed that can be used in future studies of kidney Se-containing proteins.     

Evidence that selenium in rat sperm is associated with a cysteine-rich structural protein of the mitochondrial capsules

The keratinous capsules surrounding rat sperm mitochondria were isolated 24 days after intratesticular injections of [⁷⁵Se] selenite or [³⁵S] cysteine. Dodecyl sulfate-polyacrylamide gel electrophoresis of purified, doubly labeled mitochondrial capsules revealed only a single ⁷⁵Se-labeled component, whose molecular weight was 17,000, in agreement with previously reported observations obtained with cruder sperm fractions. Most of the ³⁵S label and the major zone of stained protein on the gels coincided with the position of ⁷⁵Se, suggesting that selenium is associated with a cysteine-rich structural protein. The level of selenium in rat sperm, 195 ± 3.2 ng/10⁸ sperm (approximately 30 ppm), determined by hydride generation and atomic absorption spectrophotometry, is consistent with a structural function for this trace element in the sperm.     

Multiple selenocysteine content of selenoprotein P in rats

Partially purified selenoprotein P from rat plasma was digested with either trypsin, endoprotease Lys-C, or endoprotease Arg-C and analyzed by high pressure liquid chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Several 75Se-labeled peptides were detected. The moles of selenium in selenoprotein P were estimated based on the 75Se content of the 75Se-labeled peptide fragments. Using this method, selenoprotein P was shown to contain approximately 9 moles of selenium. This is the first report of a selenoprotein containing more than one selenium per polypeptide. These findings support the proposed function of this protein in selenium transport.     

Selenium-containing proteins of rat kidney and liver microsomes

Selenium (Se)-containing proteins in microsomal fractions of rat kidney and liver were investigated after isotopic labeling of rats with [75Se]selenite. More than 85% of the 75Se in the solubilized microsomal extracts precipitated with protein after trichloroacetic acid treatment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), used to separate the labeled protein subunits in the solubilized microsomal extracts, revealed several 75Se-containing proteins in addition to glutathione peroxidase. 75Se-labeled subunits with molecular weights of 55, 30, 26, 22, 19, and 17 kDa were present in microsomal fractions of kidney and liver. The 75Se-labeled tryptic peptide of the 55 kDa subunit had the same Rf value on a 17% SDS-PAGE gel as the peptide from plasma selenoprotein P. A time-course study of the labeling of individual protein subunits in kidney and liver microsomes from Se-supplemented and Se-deficient rats showed that most of the 75Se was associated with the 55 kDa subunit 3 hr after injection. The amount of 75Se associated with this protein subunit decreased by 12 hr, with a concurrent increase in the labeling of lower molecular-weight subunits. The results support the hypothesis that there is a mechanism for transfer of Se from the 55 kDa subunit to other Se-containing proteins.     

Selenocysteine-containing proteins from rat and monkey plasma

This investigation was carried out to determine whether a selenium-containing plasma protein in rat and monkey (Macaca mulata) plasma might be involved in selenium transport. Injection of [75Se]selenite or [75Se]selenomethionine was used to label a plasma protein. The native molecular weight of the protein from rat and monkey plasma was determined by gel filtration to be about 80 000. The molecular weight of a selenium-containing polypeptide prepared from the protein was about 45 000, as determined by gel filtration in the presence of sodium dodecyl sulfate. Selenium was attached to both the rat and monkey plasma protein in the form of the amino acid selenocysteine. The proportion of plasma selenium normally bound to the rat protein in vivo was less than 5%, and the half-life of selenium bound to the protein was a few hours. These findings are consistent with a selenium-transport function for this protein.     

Replacement of sulfide by selenide in the [4Fe-4S] clusters of the ferredoxin fromClostridium pasteurianum

The sulfur atoms of the two [4Fe-4S] clusters present in the ferredoxin from $\text{C. pasteurianum}$ have been replaced by selenium. The optical absorption spectrum of the Se-ferredoxin is slightly different from the spectrum of the native protein, but it displays the characteristic features of [4Fe-4X] ( X = S, Se) clustors. The reduced Se-ferredoxin can reduce hydrogenase, and the oxidized Se-ferredoxin can be reduced by hydrogenase in the presence of molecular hydrogen. This is the first report of sulfide substitution by selenide in an iron-sulfur protein containing [4Fe-4S] active sites.     

A selenocysteine-containing selenium-transport protein in rat plasma

A selenocysteine-containing rat plasma protein (selenoprotein P) was examined for a possible role in the transport of selenium in the rat. A time-course study of the localization of injected 75Se from [75Se]selenite indicated that one-half of the selenium was sequestered by liver tissue 1 h after injection and that one-fourth of the 75Se in the plasma was attached to selenoprotein P 3 h after injections. By 25 h there was little 75Se in plasma, and much of the 75Se had accumulated in nonhepatic tissues. 75Se was incorporated into selenoprotein P by liver slices in a process that was sensitive to the protein synthesis inhibitor cycloheximide. The fate of 75Se from intracardially injected 75Se-labeled selenoprotein P was followed in rats maintained on selenium-deficient and selenium-sufficient diets. Substantially more of the injected 75Se was present per gram wet weight in the testes and kidneys than the livers of the selenium-deprived rats after 5 h. The results indicate that selenoprotein P is synthesized in rat liver and that it transfers selenium from the liver to extrahepatic tissues.     

An external-sample liquid scintillation counting for 75Se and its application to selenoprotein detection

An external-sample liquid scintillation (LS) counting for the gamma emitter ⁷⁵Se has been developed. An expressly designed well-type LS vial and a 2,5-diphenyoxazole-1,4-bis(5-phenyl-2-oxazoyl)-benzene-xylene solution containing 35% tertrabutylzinn allow ⁷⁵Se to be counted in a standard LS counter with counting efficiency up to 43.2%, much higher than that of conventional LS counting method. This external sample LS has a good count rate linearity and exhibits low background count rates. After in vivo labeling with [⁷⁵Se]selenite, ⁷⁵Se distributions and the Se-containing proteins present in tissues of male rat were investigated by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, external-sample LS and γ-detector. Eight Se-containing proteins or protein subunits were detected to be Se-containing proteins or protein subunits in arterial wall, and their apparent molecular masses (Mr) were 76.4, 67.0, 57.4, 30.3, 25.4, 22.7, 21.7, and 15.1 kDa, respectively. In addition, eight ⁷⁵Se-labeled proteins (Mr: 66.8, 57.0, 43.1, 30.0, 24.8, 19.8, 18.0, and 14.8 kDa) were found in brain homogenates, and nine ⁷⁵Se-labeled proteins (Mr: 117.0, 78.0, 66.6, 57.2, 43.0, 38.1, 25.0, 20.1, and 18.0 kDa) were detected in testis homogenates. Some of them should be new biologically important selenoproteins that have not been identified so far.     

Ubiquity of selenium-containing tRNA in plants

Sodium[⁷⁵Se]selenite supplemented culture of Chlamydomonas, wild carrot, tobacco, bamboo, and rice cells as well as mung bean and soybean seedlings incorporated, without exception, ⁷⁵Se into tRNAs. The content of ⁷⁵Se-labeled tRNAs ranged from 0.04 to 1.89% of the total tRNAs in these seven plant species. [⁷⁵Se]tRNA samples of wild carrot and mung bean were fractionated into six or seven seleno-tRNA species by chromatography on RPC-5 column. Samples of tobacco, bamboo and Chlamydomonas each exhibited only a single seleno-tRNA species with a close interspecific resemblance in the elution position among the three samples. All these [⁷⁵Se]tRNAs contained a new, not yet identified ⁷⁵Se-labeled nucleoside, whose retention time on HPLC was distinctly different from that of the previously reported bacterial selenonucleosides. [⁷⁵Se]tRNA samples of rice, tobacco, bamboo, mung bean and Chlamydomonas also contained one or two minor ⁷⁵Se-labeled nucleosides. These results suggest that (1) selenium-containing tRNAs appear to be widespread in the plant kingdom and (2) a new, not yet characterized selenonucleoside might be universal in plants.     

Selenium increases hydrogenase expression in autotrophically cultured Bradyrhizobium japonicum and is a constituent of the purified enzyme.

We have investigated the effect of added selenite on autotrophic growth and the time course of hydrogen oxidation derepression in Bradyrhizobium japonicum 122DES cultured in a medium purified to remove selenium compounds. In addition, hydrogenase was purified to near homogeneity and examined for the specific incorporation of Se into the enzyme. The addition of Se at 0.1 microM significantly increased total cell protein and hydrogenase specific activity of harvested cells. Also, the addition of SeO3(2-) enhanced the time course of hydrogenase derepression by 133%, whereas VO3, AsO2(2-), SO2(2-), and TeO3(2-) failed to substantially affect hydrogenase derepression. During the final chromatographic purification of hydrogenase, a striking coincidence in peaks of protein content, Se radioactivity, and hydrogenase activity of fractions was obtained. The total Se content expressed per milligram of protein increased manyfold during the purification procedure. The mean Se content of the purified hydrogenase was 0.56 +/- 0.13 mol of Se per mol of enzyme. These results indicate that Se is an important element in the H2 metabolism of B. japonicum and that hydrogenase from B. japonicum is a seleno protein.     

Partial purification and characterization of the first hydrogenase isolated from a thermophilic sulfate-reducing bacterium.

A soluble [NiFe] hydrogenase has been partially purified from the obligate thermophilic sulfate-reducing bacterium Thermodesulfobacterium mobile. A 17% purification yield was obtained after four chromatographic steps and the hydrogenase presents a purity index (A398 nm/A277 nm) equal to 0.21. This protein appears to be 75% pure on SDS-gel electrophoresis showing two major bands of molecular mass around 55 and 15 kDa. This hydrogenase contains 0.6-0.7 nickel atom and 7-8 iron atoms per mole of enzyme and has a specific activity of 783 in the hydrogen uptake reaction, of 231 in the hydrogen production assay and of 84 in the deuterium-proton exchange reaction. The H2/HD ratio is lower than one in the D2-H+ exchange reaction. The enzyme is very sensitive to NO, relatively little inhibited by CO but unaffected by NO2-. The EPR spectrum of the native hydrogenase shows the presence of a [3Fe-4S] oxidized cluster and of a Ni(III) species.     

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.     

Characterization of a native subunit of the NAD-linked hydrogenase isolated from a mutant of Alcaligenes eutrophus H16

The cytoplasmic, NAD-linked hydrogenase of Alcaligenes eutrophus H16 consists of four non-identical subunits. From the mutant strain HF14, defective in this enzyme, a protein was isolated that reacted with specific antibodies raised against the wild-type hydrogenase; the reaction type was of partial identity. The same protein was also tested with specific antibodies raised against each of the four denatured subunits of the wild-type hydrogenase and was found to be completely identical with the second largest subunit; it reacted weakly with the antibody against the largest subunit and not at all with the antibody against the small subunits. In SDS-polyacrylamide gel electrophoresis the protein of the mutant migrated as a single polypeptide and corresponded to the second largest subunit of soluble hydrogenase with Mr = 56,000. The mutant enzyme strongly differed from the wild-type hydrogenase in its binding behaviour to chromatographic gels. It had pronounced hydrophobic properties and bound strongly to phenyl-Sepharose; it had high affinity to triazin dye gels. Enzymatically the polypeptide was totally inactive with NAD as electron acceptor, but showed weak residual activities with methylene blue, ferricyanide and cytochrome c. The protein also contained nickel; however, because of the instability of this polypeptide the amount varied between 0.2-1.4 nickel per enzyme molecule. As shown by ESR studies, the iron is organized in a [4Fe-4S] cluster but is partially present also in the 3Fe-form. No nickel signal could be detected. The role of the polypeptide subunit for hydrogen activation in the intact hydrogenase is discussed.     

Selenium and selenoproteins in the rat kidney

Kidney tissue contains a high concentration of selenium that is not accounted for by the known selenoprotein glutathione peroxidase (glutathione: hydrogen-peroxide oxidoreductase, EC 1.11.1.9). In order to investigate the nonglutathione peroxidase selenium, rats were isotopically labeled with [75Se]selenite over a 10-day period. After this time half of the 75Se in kidney homogenate was found in the particulate subcellular fractions. The kidney lysosomes contained unusually high levels of 75Se, yet they did not contain correspondingly high levels of glutathione peroxidase activity. Two selenoproteins having molecular weights less than 40 000 were resolved by gel filtration from a kidney supernatant fraction. A third selenoprotein exhibited a molecular weight of 75 000. This protein contained one 75 000 molecular-weight subunit, and its selenium was in the amino acid selenocysteine. The 75 000 molecular-weight protein was chromatographically distinct from glutathione peroxidase. In order to determine if these selenoproteins protect against cadmium toxicity, 109CdCl2 was administered to rats that were isotopically prelabeled with 75Se. At 3, 25 and 72 h after 109Cd administration, no 109Cd was associated with selenium-containing proteins. Two of the nonglutathione peroxidase selenoproteins were apparently unique to the kidney.     

Identification of Selenocysteine in the Proteins of Selenate-grown Vigna radiata

Selenocysteine, the selenium analog of cysteine, was identified in proteins of Vigna radiata (L.) Wilczak grown with selenate. To stabilize selenocysteine and prevent its breakdown, the carboxymethyl derivative was synthesized by the addition of iodoacetic acid to the protein extract from [⁷⁵Se]selenate-grown plants. A ⁷⁵Se-labeled component of the carboxymethylated protein hydrolysate possessed chromatographic properties identical to those of a ¹⁴C-labeled carboxymethylselenocysteine standard during paper and thin layer chromatography and during gel-exclusion, anion-exchange, and cation-exchange column chromatography. Detection of selenocysteine in proteins of a selenium-sensitive plant, and the possibility that the presence of this compound alters normal functions, provides an explanation for the toxic effects of selenium.     

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     

Escherichia coli formate-hydrogen lyase. Purification and properties of the selenium-dependent formate dehydrogenase component

The formate-hydrogen lyase complex of Escherichia coli decomposes formic acid to hydrogen and carbon dioxide under anaerobic conditions in the absence of exogenous electron acceptors. The complex consists of two separable enzymatic activities: a formate dehydrogenase and a hydrogenase. The formate dehydrogenase component (FDHH) of the formate-hydrogen lyase complex was purified to near homogeneity in two column chromatographic steps. The purified enzyme was composed of a single polypeptide of molecular weight 80,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Metal analysis showed each mole of enzyme contained 3.3 g atoms of iron. Denaturation of FDHH released a compound which, when oxidized, displayed a fluorescence spectrum similar to that of the molybdopterin cofactor found in certain other enzymes. The enzyme contained selenium in the form of selenocysteine as determined by radioactive labeling of the enzyme with 75Se and amino acid analysis. FDHH activity was maximal between pH 7.5 and 8.5; however, the enzyme was maximally stable at pH 5.3-6.4 and highly unstable above pH 7.5. Nitrate and nitrite salts caused a drastic reduction in activity. Although azide inhibited FDHH activity, it also protected the enzyme from inactivation by oxygen.     

Characterization of the nickel-iron periplasmic hydrogenase from Desulfovibrio fructosovorans

The periplasmic hydrogenase from Desulfovibrio fructosovorans grown on fructose/sulfate medium was purified to homogeneity. It exhibits a molecular mass of 88 kDa and is composed of two different subunits of 60 kDa and 28.5 kDa. The absorption spectrum of the enzyme is characteristic of an iron-sulfur protein and its absorption coefficients at 400 and 280 nm are 50 and 180 mM-1 cm-1, respectively. D. fructosovorans hydrogenase contains 11 +/- 1 iron atoms, 0.9 +/- 0.15 nickel atom and 12 +/- 1 acid-labile sulfur atoms/molecule but does not contain selenium. The amino acid composition of the protein and of its subunits, as well as the N-terminal sequences of the small and large subunits, have been determined. The cysteine residues of the protein are distributed between the large (9 residues) and the small subunits (11 residues). Electron spin resonance (ESR) properties of the enzyme are consistent with the presence of nickel(III), [3Fe-4S] and [4Fe-4S] clusters. The hydrogenase of D. fructosovorans isolated under aerobic conditions required an incubation with hydrogen or other reductants in order to express its full catalytic activity. H2 uptake and H2 evolution activities doubled after a 3-h incubation under reducing conditions. Comparison with the (NiFe) hydrogenase from D. gigas shows great structural similarities between the two proteins. However, there are significant differences between the catalytic properties of the two enzymes which can be related to the respective state of their nickel atom. ESR showed a higher proportion of the Ni-B species (g = 2.33, 2.16, 2.01) which can be related to a more facile conversion to the ready state. The periplasmic location of the enzyme and the presence of hydrogenase activity in other cellular compartments are discussed in relation to the ability of D. fructosovorans to participate actively in interspecies hydrogen transfer.     

Interactions of gold with cytosolic selenium-containing proteins in rat kidney and liver

Rats injected with aurothioglucose (ATG) for 5 days were subsequently injected with [75Se]selenious acid and killed after 3 days. Kidney and liver cytosols were chromatographed on Sephadex G-150. 75Se in kidney was associated with high molecular weight (HMW), 85,000 Mr, 26,000 Mr, and 10,000 Mr proteins and with a nonprotein fraction. The elution profile of liver cytosol was similar to that of kidney, but without a 26,000 Mr protein. ATG injection increased the association of 75Se with all fractions of kidney cytosol except the 85,000 Mr fractions, which contained Se-glutathione peroxidase (SeGSHPx) activity; 75Se in liver was increased only in HMW fractions. Unfractionated kidney cytosolic SeGSHPx activity was decreased 14% by ATG injection, but liver enzyme activity was not changed. However, Sephadex G-150 chromatography showed that total and specific activities, respectively, were decreased 28 and 23% in kidney and 25 and 16% in liver. Au coeluted with HMW and 10,000 Mr 73Se-containing kidney proteins; the latter contained 50% of the Au eluted from the column. DEAE Sephacel chromatography of the 10,000 Mr kidney protein showed that both Au and 75Se were tightly associated with metallothionein-like proteins. This study demonstrates the interaction of Au with rat liver and kidney 75Se-containing proteins.     

Selenoproteins from rat testis cytosol

Radioactive inorganic selenium, administered intraperitoneally at 1 mg/kg body weight to young adult rats, acculumulates in testes for 7 days or longer, whereas liver, kidney and serum levels fall more rapidly. 3–4 h after administration of [⁷⁵Se]selenite, 55–60% of the radioactivity in the testes was found in the cytosol, associated with protein. Ultragel ACA-22 chromatography of testis cytosol prepared 4 h after ⁷⁵Se treatment revealed a major selenoprotein having an apparent molecular weight of 59 000. Sodium dedecyl sulfate polyacrylamide gel electrophoresis indicated extensive heterogeneity of radioactivity with apparent molecular weights of about 57 000 and 45 000 and 15 000. Cytosol from rats treated 4 weeks earlier showed predominance of the 15 000 molecular weight [⁷⁵Se]selenoprotein. Sucrose density gradient ultracentrifugation at either low or high ionic strength demonstrated a single 7 S selenoprotein. Chromatography with Blue-Sepharose indicated that the radioactivity was not associated with albumin. Strong ⁷⁵Se binding to protein was demonstrated by overnight dialysis against water, 2 M NaCl, β-mercaptoethanol, 8 M urea, selenite. However, 85% of the ⁷⁵Se was removed by dialysis against 0.5 M NaOh. This stability contrasts with the lability of disulfide reagents of selenite-protein complexes formed in vitro. The fact that selenium is incorporated in substantial amounts into a discrete and stable protein suggests a physiological role for this essential trace element in the testes.