Expression of glutathione peroxidase I gene in selenium-deficient rats.

We have characterized a cDNA pGPX1211 encoding rat glutathione peroxidase I. The selenocysteine in the protein corresponded to a TGA codon in the coding region of the cDNA, similar to earlier findings in mouse and human genes, and a gene encoding the formate dehydrogenase from E. coli, another selenoenzyme. The rat GSH peroxidase I has a calculated subunit molecular weight of 22,155 daltons and shares 95% and 86% sequence homology with the mouse and human subunits, respectively. The 3'-noncoding sequence (greater than 930 bp) in pGPX1211 is much longer than that of the human sequences. We found that glutathione peroxidase I mRNA, but not the polypeptide, was expressed under nutritional stress of selenium deficiency where no glutathione peroxidase I activity can be detected. The failure of detecting any apoprotein for the glutathione peroxidase I under selenium deficiency and results published from other laboratories supports the proposal that selenium may be incorporated into the glutathione peroxidase I co-translationally.     

Subcellular distribution of selenium-containing proteins in the rat

The subcellular distribution of selenium in rat tissues was studied by measuring 75Se in animals provided for 5 months with [75Se]selenite as the main dietary source of selenium. Equilibration of the animals to a constant specific activity allowed the measurement of 75Se to be used as a specific elemental assay for selenium. Of the whole-body selenium, 51% was in the soluble fractions and 48% was bound to the particulate fractions as follows: 21% in plasma membranes, 11% in microsomes, and 16% in mitochondria. Glutathione peroxidase was primarily a soluble enzyme, but part of the activity was associated with plasma membrane in liver, mitochondria in liver and kidney, and microsomes in testes. Selenium in glutathione peroxidase accounted for about one-third of the particulate-associated selenium. These results indicate that other selenium-containing proteins besides glutathione peroxidase are present in membranes.     

Glutathione peroxidase activities from rat liver

There are two enzymes in rat liver with glutathione peroxidase activity when cumene hydroperoxide is used as substrate. One is the selenium-requiring glutathione peroxidase (glutathione:hydrogen-peroxide oxidoreductase, EC 1.11.1.9) and the other appears to be independent of dietary selenium. Activities of the two enzymes vary greatly among tissues and among animals. The molecular weight of the enzyme with selenium-independent glutathione peroxidase activity was estimated by gel filtration to be 35 000, and the subunit molecular weight was estimated by dodecyl sulfate-polyacrylamide gel electrophoresis to be 17 000. Double reciprocal plots of enzyme activity as a function of substrate concentration produced intersecting lines which are suggestive of a sequential reaction mechanism. The Km for glutathione was 0.20 mM and the Km for cumene hydroperoxide was 0.57 mM. The enzyme was inhibited by N-ethylmaleimide, but not by iodoacetic acid. Inhibition by cyanide was competitive with respect to glutathione and the Ki for cyanide was 0.95 mM. This selenium-independent glutathione peroxidase also catalyzes the conjugation of glutathione to 1-chloro-2,4-dinitrobenzene. Along with other similarities to glutathione S-transferase, this suggests that the selenium-independent glutathione peroxidase and glutathione S-transferase activities in rat liver are of the same enzyme.     

Elevation of rat liver mRNA for selenium-dependent glutathione peroxidase by selenium deficiency.

Selenium-dependent glutathione peroxidase (Se-GSH-Px, GSH-H2O2 oxidoreductase EC 1.11.1.9) is the best characterized selenoprotein in higher animals, but the mechanism whereby selenium becomes incorporated into the enzyme protein remains under investigation. To elucidate the mechanism of insertion of selenium into Ge-GSH-Px further, we have systematically analyzed and compared the results of Western blot, in vitro translation immunoprecipitation, and Northern blot experiments conducted with liver proteins and RNAs obtained from rats fed on selenium-deficient and selenium-supplemented diets. The anti-serum employed in this study was raised against an electrophoretically pure Se-GSH-Px preparation obtained from rat livers by a simplified purification procedure involving separation by high performance liquid chromatography on a hydrophobic interaction column. Different forms of Se-GSH-Px, including apo-protein, cross-reacted with this antiserum and Western blot analysis found no Se-GSH-Px protein present in livers from rats fed on selenium-deficient diets. By contrast, a distinct protein band corresponding to purified Se-GSH-Px was detected in livers from selenium-supplemented animals, a result consistent with the finding that the Se-GSH-Px activity was reduced to undetectable levels in livers of selenium-deficient rats. The in vitro translation experiments, however, indicated not only that mRNA for Se-GSH-Px was present during selenium deficiency but also that its translation products contained 2-3-fold as much immunoprecipitable protein as the products of poly(A) RNA from livers of selenium-supplemented rats. This result suggests that the Se-GSH-Px mRNA may be increased in the selenium-deficient state. Elevated levels of Se-GSH-Px mRNA were directly demonstrated in Northern blot experiments employing cDNA clone pGPX1211 as a probe. A similar increase in Se-GSH-Px mRNA was observed in such other tissues as kidney, testis, brain, and lung tissue, in selenium-deficient states. The present data support the co-translational mechanism for the incorporation of selenium into Se-GSH-Px in rat liver.     

云芝多糖对巨噬细胞GPx基因表达的影响

细胞内存在两种谷胱甘肽过氧化物酶;硒谷胱甘肽过氧化物酶和非硒谷肽甘肽过氧化物酶,它们在保护细胞免受氧化损伤等过程中起重要作用。为揭示云芝多糖作用与细胞抗氧化酶的关系,采用酶活性测定,斑点杂交等方法,探讨云芝多糖对小鼠腹腔巨噬细胞过氧化物酶表达的影响。结果显示,腹腔注射云芝多糖可以提高小鼠腹腔巨噬细胞的两种过氧化物酶活性,并使其ｍＲＮＡ含量增加,应用阻断剂的研究发现,云芝多糖对巨噬细胞ＳｅＧＰｘ及Ｇ     

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.     

Effect of selenium on the system of superoxide anion radical formation and detoxication in hepatocarcinogenesis

It is established that the introduction of selenium in combination with diethylnitrosamine into rat organisms has a preventive influence on the tumour formation. The intensity of superoxide radicals formation by the liver cell microsomes in this case decreases, while the activity of superoxide dismutase, glutathione peroxidase I, glutathione reductase and concentration of selenium in microsomes increases. The anticarcinogenic action of selenium is considered as a result of an increase in the activity of superoxide dismutase, glutathione peroxidase I and glutathione reductase. This increase induces detoxication of superoxide radicals forming in considerable amounts in rat liver cells under the effect of carcinogen.     

Some characteristics of 75Se-P,a selenoprotein found in rat liver and plasma,and comparison of it with selenoglutathione peroxidase

The selenoenzyme glutathione peroxidase cannot account for all the physiological effects of selenium in rat liver. Therefore, a study was carried out with the ultimate aim of identifying selenoproteins other than glutathione peroxidase. The incorporation of ⁷⁵Se, given as ⁷⁵SeO₃^2?, into centrifugally separated fractions of selenium-deficient and control rat livers was determined. In selenium-deficient liver much less ⁷⁵Se was incorporated into the 105,000g supernatant fraction than in controls, so this fraction was studied further by gel filtration, ion-exchange, and hydroxylapatite chromatography. Selenoglutathione peroxidase and another selenoprotein, called ⁷⁵Se-P, were separated and identified. Both these selenoproteins were also found in plasma. Selenium deficiency had opposite effects on incorporation of ⁷⁵Se by these proteins. It decreased ⁷⁵Se incorporation by glutathione peroxidase at 3 and 72 h after ⁷⁵Se injection but increased ⁷⁵Se incorporation by ⁷⁵Se-P. This suggests that ⁷⁵Se-P competes for available selenium better than does glutathione peroxidase when the element is in short supply. Apparent molecular weights of ⁷⁵Se-P from liver and plasma determined by gel filtration were, respectively, 83,000 and 79,000, which indicate proteins smaller than glutathione peroxidase. Cycloheximide pretreatment of the rat blocked ⁷⁵Se incorporation into plasma ⁷⁵Se-P. These experiments establish the existence of a selenoprotein, ⁷⁵Se-P, in rat liver and plasma which is chromatographically distinct from glutathione peroxidase and which incorporates ⁷⁵Se differently from glutathione peroxidase. ⁷⁵Se-P may account for some of the physiological effects of selenium.     

Glutathione peroxidase activities during selenium depletion of adult female rats and during selenium repletion of their offspring

The main purpose of the present investigation was to produce young rats with severe selenium deficiency, but with no clinical signs of this deficiency, and to examine their liver and red blood cell (RBC) glutathione peroxidase activities during selenium repletion. To achieve this goal, female breeders were fed a selenium-deficient diet beginning 2 weeks before mating. The liver glutathione peroxidase activity of the dams was significantly lower than the activity of comparable nonpregnant females after 5 and 10 weeks of selenium depletion. This difference arose exclusively during the period of pregnancy. In contrast, the RBC glutathione peroxidase activity was significantly increased during this period. Only traces of liver enzyme activity were found in the offspring, and the RBC enzyme activity was only 2% of that of the selenium-repleted controls. Body weight was retarded in the male offspring. However, no severe signs of clinical selenium deficiency were observed. The glutathione peroxidase activity in the liver and RBCs of the offspring was determined after 0, 2, 4, 7, 14, and approximately 40 days of selenium repletion. The liver enzyme activity increased faster in females than in males, while the opposite was found for the RBCs. After 14 days of selenium repletion, the glutathione peroxidase activity of the liver was essentially restored, and the RBC enzyme activity was about half that of the control values. This type of rat may prove useful in studies in which young selenium-deficient rats are preferable, as well as in studies of selenium functions that might not be directly related to the role of selenium in glutathione peroxidase.     

Improved procedure for X-ray photoelectron spectroscopy of selenium-glutathione peroxidase and application to the rat liver enzyme.

Purified rat liver soluble glutathione peroxidase, with a specific activity of 280 μmol of NADPH oxidized/min/mg of protein, was studied by X-ray photoelectron spectroscopy. The sampling technique developed required only 20–25 μg of protein for each sample. Selenium 3d electron signals were found in the 55.0 ± 0.3 eV region. The spectrum at the 55 eV region was free from interfering magnesium and iron. The selenium 3d electron signals observed gave evidence that selenium in glutathione peroxidase is not bound to oxygen.     

Purification and quantitation of a rat plasma selenoprotein distinct from glutathione peroxidase using monoclonal antibodies

Studies with 75Se have shown the existence of a rat plasma selenoprotein in addition to glutathione peroxidase. Because the function of the protein is not known, it has been referred to as selenoprotein P. A partially purified preparation was used to produce a monoclonal antibody to selenoprotein P. The antibody did not bind glutathione peroxidase as evidenced by its failure to remove glutathione peroxidase activity from rat plasma by immunoprecipitation. An immunoaffinity column was prepared with the monoclonal antibody, and selenoprotein P was purified 1270-fold from rat plasma in a two-step procedure. The purified selenoprotein P migrated in a single band with an Mr of 57,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoradiography demonstrated that this band contained 75Se when the protein was purified from rats which had received 75SeO2-(3). A competitive radioimmunoassay for selenoprotein P was developed. The selenoprotein P concentration in plasma of selenium-replete rats was determined with this assay to be 51 +/- 3.7 micrograms/ml. It was less than 5 micrograms/ml in plasma from selenium-deficient rats. Injection of 50 micrograms of selenium into selenium-deficient rats caused an increase in selenoprotein P from less than 10% of control to 52% of control in 6 h. Plasma glutathione peroxidase activity increased only from 2.2 to 3.1% of control. These experiments demonstrate that rat plasma contains a selenoprotein distinct from glutathione peroxidase. The concentration of this selenoprotein is depressed in selenium deficiency, as is glutathione peroxidase activity, but selenoprotein P increases more rapidly when selenium is supplied than does glutathione peroxidase activity.     

Expression of a cDNA encoding a rat liver glutathione S-transferase Ya subunit in Escherichia coli

A full length cDNA clone, pGTB38 (C. B. Pickett et al. (1984) J. Biol. Chem. 259, 5182-5188), complementary to a rat liver glutathione S-transferase Ya mRNA has been expressed in Escherichia coli. The cDNA insert was isolated from pGTB38 using MaeI endonuclease digestion and was inserted into the expression vector pKK2.7 under the control of the tac promoter. Upon transformation of the expression vector into E. coli, two protein bands with molecular weights lower than the full-length Ya subunit were detected by Western blot analysis in the cell lysate of E. coli. These lower-molecular-weight proteins most likely result from incorrect initiation of translation at internal AUG codons instead of the first AUG codon of the mRNA. In order to eliminate the problem of incorrect initiation, the glutathione S-transferase Ya cDNA was isolated from the expression vector and digested with Bal31 to remove extra nucleotides from the 5' noncoding region. The protein expressed by this expression plasmid, pKK-GTB34, comigrated with the Ya subunit on sodium dodecyl sulfate polyacrylamide gels and was recognized by antibodies against the YaYc heterodimer. The expressed Ya homodimer was purified by S-hexylglutathione affinity and ion-exchange chromatographies. Approximately 50 mg pure protein was obtained from 9 liters of E. coli culture. The expressed Ya homodimer displayed glutathione-conjugating, peroxidase, and isomerase activities, which are identical to those of the native enzyme purified from rat liver cytosol. Protein sequencing indicates that the expressed protein has a serine as the NH2 terminus whereas the NH2 terminus of the glutathione S-transferase Ya homodimer purified from rat liver cytosol is apparently blocked.     

Differential regulation of rat liver selenoprotein mRNAs in selenium deficiency.

Selenium deficiency causes a fall in the concentrations of selenoproteins but selenoprotein P and type I iodothyronine 5'-deiodinase (5'-deiodinase) are more resistant to this effect than is glutathione peroxidase. To investigate the differential regulation of these selenoproteins, a selenium-deficient diet was fed to weanling rats for 14.5 weeks and their hepatic mRNAs were measured by Northern analysis. Levels of all 3 mRNAs fell progressively with time. Selenoprotein P and 5'-deiodinase mRNAs remained higher at all time points relative to control than glutathione peroxidase mRNA. mRNA decreases were mirrored by decreases in glutathione peroxidase activity and selenoprotein P concentration. However, the decreases in the protein levels were greater than the decreases in their mRNAs, suggesting that synthesis of both proteins was limited to a similar extent at the translational level by the availability of selenium. In addition to this apparently unregulated translational effect, these results point to a pretranslational regulation, affecting mRNA levels, which could account for the differential effect of selenium deficiency on glutathione peroxidase and the other selenoproteins. This regulation might serve to direct selenium to selenoprotein P and 5'-deiodinase when limited amounts of the element are available.     

Glutathione peroxidase activity in selenium-deficient rat liver

Glutathione peroxidase activity in the liver supernatant from rats fed a Se-deficient diet for 2 weeks was 8% of control when measured with H₂O₂ but 42% of control when assayed with cumene hydroperoxide. Two peaks of glutathione peroxidase activity were present in the Sephadex G-150 gel filtration chromatogram of rat liver supernatant when 1.5 mM cumene hydroperoxide was used as substrate. Only the first peak was detected when 0.25 mM H₂O₂ was used as substrate. The first peak was absent from chromatograms of Se-deficient rat liver supernatants; but the second peak, which eluted at a position corresponding to M.W. = 39,000, appeared unchanged. The second peak thus represents a second glutathione peroxidase activity which catalyzes the destruction of organic hydroperoxides but has little activity toward H₂O₂ and which persists in severe selenium deficiency.     

The role of selenium in the regulation of lipid peroxidation in biological membranes and glutathione peroxidase activity

The antioxidative effect of selenium cannot be exclusively due to the functioning of the selenium-dependent glutathione peroxidase mechanism of utilization of various hydroperoxides. This hypothesis is based on the following experimental evidence. Selenium ions are readily incorporated into animal organs and tissues immediately after injection (2 hours) as well as into cell organelles and cytosol where they inhibit lipid peroxidation. The activity of glutathione peroxidase (EC 1.1.1.19) in rat liver and guinea pig cytosol is thereby unchanged but increases drastically after 12 hours reaching a maximum an the 3rd-4th day. The effectiveness of lipid peroxidation inhibition does not increase under these conditions. Although the glutathione peroxidase activity is absent in the nuclei and microsomes, exogenous selenium inhibits lipid peroxidation in these organelles. The activity of the rat liver cytosolic enzyme markedly exceeds that of its guinea pig counterpart. However, lipid peroxidation in guinea pig liver occurs less intensively than that in rat liver cytosol.     

Isolation and characterization of a DNA sequence complementary to rat liver glutathione S-transferase B mRNA

Total rat liver poly(A+)-RNA has been isolated from phenobarbital-treated rats and fractionated on sucrose gradients to enrich for glutathione S-transferase B mRNA. Poly(A+)-RNA fractions were assayed for glutathione S-transferase B mRNA activity by in vitro translation and those fractions enriched in glutathione S-transferase B mRNA were used as a template for cDNA synthesis. The cDNA was cloned into the PstI site of pBR322 by G-C tailing. Bacterial clones harboring inserts complementary to glutathione S-transferase mRNA were identified by colony hybridization using a [32P]cDNA probe reverse transcribed from poly(A+)-RNA enriched significantly in glutathione S-transferase B mRNA and by hybrid-select translation. Two recombinant clones, pGTB6 and pGTB15 hybrid-selected the mRNAs specific for the Ya and Yc subunits, indicating these two mRNAs share significant sequence homology. Radiolabeled pGTB6 was utilized in RNA gel-blot experiments to determine that the size of glutathione S-transferase B mRNA is 980 nucleotides and the degree of induction of the mRNA in response to 3-methylcholanthrene administration is threefold.     

Molecular biology of selenium with implications for its metabolism.

Selenium has a highly specific metabolism centered around its incorporation as selenocysteine into selenoproteins. An outline of this metabolism has emerged from recent molecular biological and biochemical studies of bacteria and animals. A unique tRNA, designated tRNA[Ser]Sec, is charged with L-serine, which is then converted through at least two steps to selenocysteine. With the aid of a unique translation factor, the selenocysteinyl-tRNA[Ser]Sec recognizes specific UGA codons in mRNA to insert selenocysteine into the primary structure of selenoproteins. Turnover of selenoproteins presumably liberates selenocysteine which is toxic in its free form. Selenocysteine beta-lyase catabolizes free selenocysteine and makes its selenium available for reuse. Proteins contain almost all the selenium in animals. Of the known selenoproteins, the glutathione peroxidases contain the most selenium. Cellular and plasma glutathione peroxidases are products of different genes but have 44% identity of amino acid sequence. There is evidence for other proteins of this family. Selenoprotein P is an unrelated protein with multiple selenocysteines in its primary structure. It contains most of the selenium in rat plasma. Studies of the regulation of cellular glutathione peroxidase by selenium have yielded conflicting results, but there is a strong suggestion that mRNA levels of the rodent liver glutathione peroxidase decrease in selenium deficiency. This could be a mechanism for directing selenium to the synthesis of other selenoproteins. Although present knowledge allows construction of an outline of selenium metabolism, several steps have not been characterized and little is known about mechanisms of its regulation.