ABSTRACT
The selenoenzyme thioredoxin reductase (TR) can recycle ascorbic acid, which in turn can recycle alpha-tocopherol. Therefore, we evaluated the role of selenium in ascorbic acid recycling and in protection against oxidant-induced loss of alpha-tocopherol in cultured liver cells. Treatment of HepG2 or H4IIE cultured liver cells for 48 h with sodium selenite (0-116 nmol/l) tripled the activity of the selenoenzyme TR, measured as aurothioglucose-sensitive dehydroascorbic acid (DHA) reduction. However, selenium did not increase the ability of H4IIE cells to take up and reduce 2 mM DHA, despite a 25% increase in ascorbate-dependent ferricyanide reduction (which reflects cellular ascorbate recycling). Nonetheless, selenium supplements both spared ascorbate in overnight cultures of H4IIE cells, and prevented loss of cellular alpha-tocopherol in response to an oxidant stress induced by either ferricyanide or diazobenzene sulfonate. Whereas TR contributes little to ascorbate recycling in H4IIE cells, selenium spares ascorbate in culture and alpha-tocopherol in response to an oxidant stress.
Subject(s)
Ascorbic Acid/metabolism , Hepatocytes/metabolism , Oxidative Stress , Sodium Selenite/pharmacology , Thioredoxin-Disulfide Reductase/metabolism , alpha-Tocopherol/metabolism , Animals , Culture Media , Dehydroascorbic Acid/metabolism , Diazonium Compounds/pharmacology , Ferricyanides/metabolism , Ferricyanides/pharmacology , Hepatocytes/drug effects , Humans , Oxidation-Reduction , Oxidoreductases/metabolism , Rats , Selenocysteine/pharmacology , Sulfanilic Acids/pharmacology , Thioredoxins/pharmacology , Tumor Cells, CulturedABSTRACT
BACKGROUND: Oxidative stress is pivotal in atherogenesis. Although the most widely used indirect assay to quantify oxidative stress is LDL oxidative susceptibility, direct assays such as urinary F(2)-isoprostanes have shown great promise. METHODS: We evaluated the utility of both a direct measure of oxidative stress (urinary F(2)-isoprostanes) and an indirect measure of copper-catalyzed, LDL oxidation in a model of increased oxidative stress (diabetes). We also evaluated an enzyme immunoassay (EIA) method for urinary F(2)-isoprostanes with a gas chromatography-mass spectrometry method. RESULTS: Excellent intraassay and interassay CVs of <4% were obtained with our EIA method. A good correlation was obtained between the two methods (r = 0.80; n = 68) of F(2)-isoprostane measurement. An excellent correlation for F(2)-isoprostane concentrations was obtained between a timed collection vs 24-h urine (r = 0.96; n = 46). Baseline F(2)-isoprostane concentrations by EIA were significantly higher in both type 2 diabetics with and without macrovascular complications compared with controls (P <0.001). Supplementation with alpha-tocopherol led to a significant reduction in F(2)-isoprostane concentrations in all diabetic patients compared with baseline values (2.51 +/- 1.76 compared with 1.69 +/- 1.32 ng/mg creatinine; P <0.001). There were no significant differences in LDL oxidation in both diabetic groups compared with controls. alpha-Tocopherol supplementation led to significant increases in the lag phase of oxidation as measured by 3 indices in all groups. CONCLUSIONS: The measurement of urinary F(2)-isoprostanes provides a direct measure of in vivo lipid peroxidation and oxidative stress and appears to be superior to an indirect measure, e.g., LDL oxidative susceptibility, in type 2 diabetes.
Subject(s)
Diabetes Mellitus, Type 2/metabolism , Dinoprost/urine , Lipoproteins, LDL/metabolism , Oxidative Stress , Diabetes Mellitus, Type 2/urine , Dinoprost/analogs & derivatives , F2-Isoprostanes , Female , Gas Chromatography-Mass Spectrometry , Humans , Immunoenzyme Techniques , Male , Oxidation-Reduction , Reproducibility of ResultsABSTRACT
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.
Subject(s)
Proteins/metabolism , Selenium Compounds/pharmacokinetics , Selenium/blood , Selenomethionine/pharmacokinetics , Adult , Animals , Humans , Kinetics , Male , Rats , Selenic Acid , SelenoproteinsABSTRACT
Selenium and vitamin E deficiencies were studied as part of an evaluation of oxidant defenses in guinea pigs. Male guinea pigs (100-120 g) were fed a control diet (C) or the diet without selenium (0 Se), without vitamin E (0 E), or without either selenium or vitamin E (0 Se-0 E). Between d 30 and 35, 7 of 13 guinea pigs fed the 0 Se-0 E diet were euthanized because of severe weakness of their extremities. No guinea pigs in the other diet groups developed weakness. Guinea pigs from each group were killed on d 37. Selenium deficiency and vitamin E deficiency were verified by measurement of glutathione peroxidase and alpha-tocopherol. Creatine phophokinase (CPK) activity was greater than controls in both groups fed vitamin E-deficient diets, but the increase was greater in the 0 Se-0 E group than in the 0 E group. Muscle F(2)-isoprostanes were greater than controls in both groups fed vitamin E-deficient diets with the level in the 0 Se-0 E group greater than that in the 0 E group. Histologic muscle necrosis was severe in the 0 Se-0 E group, minimal in the 0 E group and absent from other groups. The diets used in this study induced selenium and vitamin E deficiencies in guinea pigs. The study demonstrates that combined selenium and vitamin E deficiency results in a fatal myopathy in guinea pigs that is associated with lipid peroxidation in the affected muscle. This nutritional myopathy is much more severe than the myopathy that occurs with vitamin E deficiency alone.
Subject(s)
Muscle, Skeletal/physiopathology , Muscular Diseases/etiology , Selenium/deficiency , Vitamin E Deficiency/metabolism , Animals , Body Weight , Creatine Kinase/analysis , Creatine Kinase/blood , Diet , Dinoprost/analogs & derivatives , Dinoprost/analysis , F2-Isoprostanes , Guinea Pigs , Liver/metabolism , Male , Muscular Diseases/blood , Muscular Diseases/physiopathology , Necrosis , Survival Analysis , Vitamin E/analysis , Vitamin E/blood , Vitamin E Deficiency/bloodABSTRACT
Selenocysteine (Sec) tRNA (tRNA([Ser]Sec)) serves as both the site of Sec biosynthesis and the adapter molecule for donation of this amino acid to protein. The consequences on selenoprotein biosynthesis of overexpressing either the wild type or a mutant tRNA([Ser]Sec) lacking the modified base, isopentenyladenosine, in its anticodon loop were examined by introducing multiple copies of the corresponding tRNA([Ser]Sec) genes into the mouse genome. Overexpression of wild-type tRNA([Ser]Sec) did not affect selenoprotein synthesis. In contrast, the levels of numerous selenoproteins decreased in mice expressing isopentenyladenosine-deficient (i(6)A(-)) tRNA([Ser]Sec) in a protein- and tissue-specific manner. Cytosolic glutathione peroxidase and mitochondrial thioredoxin reductase 3 were the most and least affected selenoproteins, while selenoprotein expression was most and least affected in the liver and testes, respectively. The defect in selenoprotein expression occurred at translation, since selenoprotein mRNA levels were largely unaffected. Analysis of the tRNA([Ser]Sec) population showed that expression of i(6)A(-) tRNA([Ser]Sec) altered the distribution of the two major isoforms, whereby the maturation of tRNA([Ser]Sec) by methylation of the nucleoside in the wobble position was repressed. The data suggest that the levels of i(6)A(-) tRNA([Ser]Sec) and wild-type tRNA([Ser]Sec) are regulated independently and that the amount of wild-type tRNA([Ser]Sec) is determined, at least in part, by a feedback mechanism governed by the level of the tRNA([Ser]Sec) population. This study marks the first example of transgenic mice engineered to contain functional tRNA transgenes and suggests that i(6)A(-) tRNA([Ser]Sec) transgenic mice will be useful in assessing the biological roles of selenoproteins.
Subject(s)
Protein Biosynthesis , Proteins , RNA, Transfer, Amino Acid-Specific/biosynthesis , Animals , Base Sequence , Blotting, Northern/methods , Gene Expression , Isopentenyladenosine/genetics , Isopentenyladenosine/metabolism , Mice , Mice, Transgenic , Molecular Sequence Data , Nucleic Acid Conformation , Selenium/metabolism , SelenoproteinsABSTRACT
Low doses of diquat cause massive liver necrosis and death of selenium-deficient rats within a few hours. Protection against this injury by selenium correlates with the presence of selenoprotein P, an extracellular selenoprotein that associates with endothelial cells. Selenium-deficient rats were injected with diquat (10 mg/kg) and their livers were removed for light and electron microscopy at times up to 120 minutes after injection. Selenium-replete animals were studied before and 120 minutes after the same dose of diquat. With selenium deficiency, diquat caused injury to centrilobular endothelial cells. This injury was evident 20 minutes after diquat injection and progressed to cell loss at 60 minutes after diquat injection. At 120 minutes, endothelial cells were virtually absent from the centrilobular regions and hepatocytes in those areas were undergoing necrosis. Portal and midzonal areas remained normal in selenium-deficient livers, as did the entire liver lobule of selenium-replete rats. These findings indicate that the initial liver lesion in selenium-deficient rats given diquat is injury of the endothelial cells in the centrilobular region. After detachment of the endothelial cells, centrilobular hepatocytes undergo necrosis. We postulate that selenoprotein P protects the centrilobular endothelial cells against injury by oxidant molecules that result from diquat administration.
Subject(s)
Diquat/toxicity , Liver/drug effects , Selenium/deficiency , Animals , Endothelium/drug effects , Endothelium/pathology , Endothelium/ultrastructure , Glutathione Peroxidase/blood , Liver/pathology , Liver/ultrastructure , Male , Proteins/metabolism , Rats , Rats, Sprague-Dawley , Reference Values , Selenium/pharmacology , Selenoprotein P , SelenoproteinsABSTRACT
Selenoprotein P is an extracellular protein that has been postulated to have an oxidant defense function. It has survival-promoting properties for cultured neurons and its mRNA is present in the brain. This study sought to determine the primary structure of rat brain selenoprotein P and to assess its production by cultured brain cells. The cDNA of selenoprotein P was isolated from a rat brain cDNA library and was found to encode the same peptide sequence as rat liver cDNA. Thus the primary structure of brain selenoprotein P is the same as selenoprotein P from liver. Astrocytes and a cerebellar granule cell preparation (CGC) were obtained from rat brains and established in culture. The CGC was estimated to contain up to 5% glial cells. Both preparations were shown to contain selenoprotein P mRNA. During incubation with (75)Se-labeled selenite, both cell preparations secreted a (75)Se-labeled protein into the medium that corresponded in size to selenoprotein P. Also, the (75)Se-labeled protein could be precipitated from both media with an antiserum to selenoprotein P. This shows that astrocytes and the CGC secrete selenoprotein P. Selenoprotein P is made in the brain and may have an oxidant defense function there.
Subject(s)
Astrocytes/metabolism , Protein Biosynthesis , Animals , Autoradiography , Base Sequence , Cells, Cultured , Cloning, Molecular , DNA, Complementary/isolation & purification , Electrophoresis, Polyacrylamide Gel , Gene Library , Molecular Sequence Data , Proteins/genetics , Proteins/metabolism , RNA, Messenger/biosynthesis , Rats , Selenium Radioisotopes , Selenoprotein P , SelenoproteinsABSTRACT
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.
Subject(s)
Immunoglobulin G/chemistry , Nitrates/pharmacology , Oxidants/pharmacology , Selenium/pharmacokinetics , Selenomethionine , Serum Albumin/chemistry , Adult , Humans , Immunoglobulin G/drug effects , Male , Oxidation-Reduction , Serum Albumin/drug effectsABSTRACT
Selenoproteins contain selenium in stoichiometric amounts. Most are synthesized by a process that decodes UGA codons as selenocysteine. Twelve animal selenoproteins have been characterized, and biochemical functions have been described for all but three. Two of these "orphan" selenoproteins are discussed in this paper. Selenoprotein P is an extracellular glycoprotein that contains multiple selenocysteines. It binds heparin and associates with endothelial cells. Two isoforms have been identified. Plasma concentration of selenoprotein P correlates with protection against diquat liver injury, suggesting that the protein protects against oxidant injury. Selenoprotein W is a small intracellular protein that contains one selenocysteine. It binds glutathione and has been suggested to function in oxidant defense. The postulated oxidant defense properties of these selenoproteins are consistent with the facile thiol-redox properties of selenocysteine. It can be predicted that more proteins will be discovered that take advantage of the chemical properties of selenium.
Subject(s)
Proteins/physiology , Amino Acid Sequence , Animals , Molecular Sequence Data , Protein Biosynthesis , Selenium , Selenoprotein P , Selenoprotein W , SelenoproteinsABSTRACT
Recycling of ascorbic acid from its oxidized forms is required to maintain intracellular stores of the vitamin in most cells. Since the ubiquitous selenoenzyme thioredoxin reductase can recycle dehydroascorbic acid to ascorbate, we investigated the possibility that the enzyme can also reduce the one-electron-oxidized ascorbyl free radical to ascorbate. Purified rat liver thioredoxin reductase catalyzed the disappearance of NADPH in the presence of low micromolar concentrations of the ascorbyl free radical that were generated from ascorbate by ascorbate oxidase, and this effect was markedly stimulated by selenocystine. Dehydroascorbic acid is generated by dismutation of the ascorbyl free radical, and thioredoxin reductase can reduce dehydroascorbic acid to ascorbate. However, control studies showed that the amounts of dehydroascorbic acid generated under the assay conditions used were too low to account for the observed loss of NADPH. Electron paramagnetic resonance spectroscopy directly confirmed that the reductase decreased steady-state ascorbyl free radical concentrations, as expected if thioredoxin reductase reduces the ascorbyl free radical. Dialyzed cytosol from rat liver homogenates also catalyzed NADPH-dependent reduction of the ascorbyl free radical. Specificity for thioredoxin reductase was indicated by loss of activity in dialyzed cytosol prepared from livers of selenium-deficient rats, by inhibition with aurothioglucose at concentrations selective for thioredoxin reductase, and by stimulation with selenocystine. Microsomal fractions prepared from rat liver showed substantial NADH-dependent ascorbyl free radical reduction that was not sensitive to selenium depletion. These results suggest that thioredoxin reductase can function as a cytosolic ascorbyl free radical reductase that may complement cellular ascorbate recycling by membrane-bound NADH-dependent reductases.
Subject(s)
Dehydroascorbic Acid/analogs & derivatives , Metalloproteins/metabolism , Selenium , Thioredoxin-Disulfide Reductase/metabolism , Animals , Aurothioglucose/pharmacology , Cystine/analogs & derivatives , Cystine/pharmacology , Cytosol/metabolism , Dehydroascorbic Acid/metabolism , Electron Spin Resonance Spectroscopy , Free Radicals , Liver/enzymology , Metalloproteins/antagonists & inhibitors , Metalloproteins/drug effects , Microsomes, Liver/enzymology , NADP/metabolism , Organoselenium Compounds/pharmacology , Oxidation-Reduction , Rats , Selenium/deficiency , Thioredoxin-Disulfide Reductase/antagonists & inhibitors , Thioredoxin-Disulfide Reductase/drug effectsABSTRACT
Plasma selenium concentration is decreased in patients with cirrhosis and, based on this finding, it has been suggested that patients with cirrhosis are selenium deficient. We measured plasma selenium concentration and the two plasma selenoproteins, glutathione peroxidase (GSHPx-3) and selenoprotein P, in the plasma of patients with cirrhosis of Child classes A, B, and C and in control subjects. Plasma selenium declined in proportion to the severity of the cirrhotic condition, as indicated by the Child class. Selenoprotein P, which originates largely in the liver, declined in a similar manner. Plasma glutathione peroxidase activity increased, and GSHPx-3 originates in the kidney. Selenium in the non-selenoprotein pool, shown by others to be largely selenomethionine in albumin, declined. Thus, although plasma selenium is decreased in patients with cirrhosis, the increase in plasma glutathione peroxidase activity, which occurs in them, suggests that patients with cirrhosis do not have selenium deficiency.
Subject(s)
Liver Cirrhosis/blood , Selenium/blood , Adult , Aged , Female , Humans , Male , Middle Aged , Selenium/deficiencyABSTRACT
We reported previously that the selenium status of rats influences both the steady-state levels and distributions of two selenocysteine tRNA isoacceptors and that these isoacceptors differ by a single methyl group attached to the ribosyl moiety at position 34. In this study, we demonstrate that repletion of selenium-deficient rats results in a gradual, tissue-dependent shift in the distribution of these isoacceptors. Rats fed a selenium-deficient diet possess a greater abundance of the species unmethylated on the ribosyl moiety at position 34 compared to the form methylated at this position. A redistribution of the Sec-tRNA isoacceptors occurred in tissues of selenium-supplemented rats whereby the unmethylated form gradually shifted toward the methylated form. This was true in each of four tissues examined, muscle, kidney, liver and heart, although the rate of redistribution was tissue-specific. Muscle manifested a predominance of two minor serine isoacceptors under conditions of extreme selenium-deficiency which also appeared to respond to selenium. Ribosomal binding studies revealed that one of the two additional isoacceptors decodes the serine codeword, AGU, and the second decodes the serine codeword, UCU. Interestingly, muscle and heart were the slower tissues to return to a 'selenium adequate' tRNA distribution pattern.
Subject(s)
RNA, Transfer, Amino Acid-Specific/metabolism , Selenium/deficiency , Selenium/metabolism , Animals , Chromatography, Ion Exchange , Codon/genetics , Diet , Kidney/metabolism , Liver/metabolism , Male , Muscles/metabolism , Myocardium/metabolism , Organ Specificity , Proteins/metabolism , RNA, Transfer, Ser/metabolism , Rats , Rats, Sprague-Dawley , Ribosomes/metabolism , Selenium/administration & dosage , SelenoproteinsSubject(s)
Liver/enzymology , Selenium/deficiency , Thioredoxin-Disulfide Reductase/metabolism , Animals , Aurothioglucose , Cytosol/enzymology , Dithionitrobenzoic Acid , Indicators and Reagents , Kinetics , Oxidation-Reduction , Rats , Reference Values , Reproducibility of Results , Sensitivity and Specificity , Spectrophotometry/methods , Thioredoxin-Disulfide Reductase/analysisABSTRACT
Purification of selenoprotein P from rat plasma has allowed detailed characterization of the protein from that species. Chromatographic studies have revealed the existence of at least two isoforms of the protein. One isoform is a truncated protein with termination of protein synthesis occurring at the second selenocysteine codon. Immunohistochemical studies have shown that the rat protein is associated with capillary endothelial cells in the liver, kidney, and brain. In vivo experiments were designed to study the relationship between selenoprotein P and lipid peroxidation. F2 isoprostanes were measured to quantitate the extent of lipid peroxidation caused by diquat. Using selenium-deficient rats supplemented with selenium, it was demonstrated that selenoprotein P appearance correlated with disappearance of diquat-induced lipid peroxidation. This finding is consistent with the protein serving to protect the plasma membrane from oxidative damage. Development of a radioimmunoassay to measure selenoprotein P has allowed assessment of the protein in humans. Measurements of the protein in selenium-deficient Chinese subjects indicated that it can be used as an index of selenium nutritional status in humans.
Subject(s)
Proteins/metabolism , Animals , Humans , Immunochemistry , Nutritional Status , Rats , Selenium/administration & dosage , Selenium/deficiency , Selenoprotein P , SelenoproteinsABSTRACT
Recycling of ascorbate from its oxidized forms is essential to maintain stores of the vitamin in human cells. Whereas reduction of dehydroascorbate to ascorbate is thought to be largely GSH-dependent, we reconsidered the possibility that the selenium-dependent thioredoxin system might contribute to ascorbate regeneration. We found that purified rat liver thioredoxin reductase functions as an NADPH-dependent dehydroascorbate reductase, with an apparent Km of 2. 5 mM for dehydroascorbate, and a kcat of 90 min-1. Addition of 2.8 microM purified rat liver thioredoxin lowered the apparent Km to 0.7 mM, without affecting the turnover (kcat of 71 min-1). Since thioredoxin reductase requires selenium, we tested the physiologic importance of this enzyme for dehydroascorbate reduction in livers from control and selenium-deficient rats. Selenium deficiency lowered liver thioredoxin reductase activity by 88%, glutathione peroxidase activity by 99%, and ascorbate content by 33%, but did not affect GSH content. NADPH-dependent dehydroascorbate reductase activity due to thioredoxin reductase, on the basis of inhibition by aurothioglucose, was decreased 88% in dialyzed liver cytosolic fractions from selenium-deficient rats. GSH-dependent dehydroascorbate reductase activity in liver cytosol was variable, but typically 2-3-fold that of NADPH-dependent activity. These results show that the thioredoxin system can reduce dehydroascorbate, and that this function is required for maintenance of liver ascorbate content.
Subject(s)
Ascorbic Acid/biosynthesis , Dehydroascorbic Acid/metabolism , Thioredoxin-Disulfide Reductase/metabolism , Animals , Kinetics , Liver/enzymology , Liver/metabolism , NADP/metabolism , Rats , Selenium/deficiency , Thioredoxins/metabolismABSTRACT
Selenoprotein P is an extracellular heparin-binding protein that has been implicated in protecting the liver against oxidant injury. Its location in liver, kidney, and brain was determined by conventional immunohistochemistry and confocal microscopy using a polyclonal antiserum. Selenoprotein P is associated with endothelial cells in the liver and is more abundant in central regions than in portal regions. It is also present in kidney glomeruli associated with capillary endothelial cells. Staining of selenoprotein P in the brain is also confined to vascular endothelial cells. The heparin-binding properties of selenoprotein P could be the basis for its binding to tissue. Its localization to the vicinity of endothelial cells is potentially relevant to its oxidant defense function.
Subject(s)
Brain/metabolism , Endothelium/metabolism , Kidney/metabolism , Liver/metabolism , Proteins/metabolism , Selenium , Animals , Brain/cytology , Endothelium/cytology , Immunohistochemistry , Kidney/cytology , Liver/cytology , Male , Microscopy, Confocal , Oxidation-Reduction , Rats , Rats, Sprague-Dawley , Selenoprotein P , SelenoproteinsABSTRACT
Animal thioredoxin reductase is a selenoprotein. In this study, thioredoxin reductase activities in liver, kidney, and brain have been compared in rats fed selenium-deficient and control diets for 14 weeks following weaning. Selenium deficiency caused a decrease in thioredoxin reductase activity from control to 4.5% in liver and 11% in kidney. However, brain thioredoxin reductase activity was not affected by selenium deficiency of this severity. Gold inhibited thioredoxin reductase activity in the liver in a manner typical of its effect on selenoenzymes. Repletion of selenium-deficient rats with injections of selenium caused thioredoxin reductase activity to increase more rapidly in the liver than glutathione peroxidase activity but more slowly than selenoprotein P. These results indicate that thioredoxin reductase activity in liver and kidney is sensitive to selenium nutritional status but that brain thioredoxin reductase activity is less sensitive.
Subject(s)
Selenium/deficiency , Thioredoxin-Disulfide Reductase/metabolism , Animals , Aurothioglucose/pharmacology , Brain/enzymology , Diet , Enzyme Inhibitors/pharmacology , Glutathione Peroxidase/metabolism , In Vitro Techniques , Kidney/enzymology , Liver/drug effects , Liver/enzymology , Liver/metabolism , Male , Nutritional Status , Proteins/metabolism , Rats , Rats, Sprague-Dawley , Selenium/administration & dosage , Selenoprotein P , Selenoproteins , Thioredoxin-Disulfide Reductase/antagonists & inhibitors , Thioredoxin-Disulfide Reductase/deficiencyABSTRACT
Selenoprotein P and glutathione peroxidase are selenoproteins that are synthesized by hepatocytes. The production of these selenoproteins by human and rat liver cell lines has been assessed at several levels of selenium supplementation and compared with one another. HepG2 and H4IIE cells were cultured in serum-free medium without selenium supplementation for 48 h; then sodium selenite was added to the medium to give final concentrations of 0, 1, 2.5, 5, or 10 ng selenium/ml medium. After 48 h, selenoprotein P concentration in the medium, cellular glutathione peroxidase activity, and the mRNA levels of the two selenoproteins were determined. Selenium deficiency caused a decrease in selenoprotein mRNA and protein levels. The extent of decrease depended on the cell line examined. In selenium-deprived HepG2 cells, selenoprotein P release decreased to 10% of the release by selenium-replete cells. Under the same conditions, cellular glutathione peroxidase activity decreased to 33%. H4IIE cells showed the opposite results with cellular glutathione peroxidase activity decreasing to 13% and selenoprotein P release decreasing to 40% of selenium-replete cells. The effect of dithiothreitol on secretion of selenoprotein P by H4IIE cells was examined. Selenoprotein P secretion was inhibited by dithiothreitol, suggesting that disulfide bond formation is necessary for secretion of the mature protein.
Subject(s)
Glutathione Peroxidase/metabolism , Liver/metabolism , Proteins/metabolism , Selenium/metabolism , Animals , Cycloheximide/pharmacology , Humans , Protein Synthesis Inhibitors/pharmacology , Rats , Selenoprotein P , Selenoproteins , Tumor Cells, CulturedABSTRACT
Several forms of selenoprotein P that share the same N-terminal sequence have been identified in rat plasma, but only one selenoprotein P mRNA has been characterized. The open reading frame of the mRNA contains 10 UGAs that presumably code for selenocysteine residues. Using heparin-Sepharose, we isolated two of the protein forms from immunoaffinity-purified selenoprotein P. One of the forms, Se-P45B, migrates at 45 kDa on SDS-polyacrylamide gel electrophoresis, and the other, Se-P57B, migrates at 57 kDa. These two forms were cleaved with cyanogen bromide, and both yielded 40-kDa fragments that were consistent with those fragments being an inter-methionine peptide near the N terminus of the predicted polypeptide. A 20-kDa fragment present in the cleavage products of Se-P57B was absent from the products of Se-P45B. This result suggested that Se-P45B lacks the C-terminal region of the predicted polypeptide. Carboxypeptidase P digestion of Se-P45B indicated that its C-terminal amino acid is Ser244, the amino acid immediately upstream from the predicted second selenocysteine. C-terminal analysis of Se-P57B indicated that its final residue is Asn366, the last amino acid predicted by the cDNA sequence. Amino acid composition analyses of the two forms were consistent with both arising from the same mRNA. Immunoaffinity-purified selenoprotein P was digested with proteases, and the resulting peptides were separated and sequenced. Only amino acid sequences predicted by the cDNA were found, and 80% of the predicted amino acid sequence was confirmed. These results are compatible with Se-P45B arising from termination of translation at the second in-frame UGA codon and all of the 10 in-frame UGA codons being read through to produce Se-P57B. These findings demonstrate that selenoprotein P isoforms of differing peptide lengths are present in plasma. They raise the possibility that the second UGA codon in selenoprotein P mRNA can have alternative functions: coding for the incorporation of selenocysteine or coding for termination of translation.
Subject(s)
Proteins/chemistry , Amino Acid Sequence , Animals , Molecular Sequence Data , Molecular Weight , Peptide Chain Termination, Translational , Peptide Fragments/chemistry , Peptide Mapping , Rats , Selenium , Selenoprotein P , SelenoproteinsABSTRACT
SDS-PAGE of immunoaffinity-purified rat seleno-protein P demonstrates a major band at 57 kDa and a less intense band at 45 kDa, suggesting the existence of more than one form of the protein. Separate experiments were carried out in which plasma from rats administered 75Se and immunoaffinity-purified 75Se-labeled selenoprotein P were applied to a heparin-Sepharose column at pH 7. In each experiment three peaks of 75Se-labeled protein were eluted during a continuous gradient from pH 7 to pH 8.5. The remaining 75Se-labeled material was eluted as a single peak by 1 M NaCl. Phosphorimaging of the SDS-PAGE gel of the peaks revealed that the first peak (1a) contained two bands (45 and 57 kDa) while the three remaining peaks each contained one band (peak 1b, 45 kDa and peaks 2 and 3, 57 kDa). N-terminal sequencing of the first eight amino acids of each band revealed that all five have the same N-terminal amino acid sequence and therefore are forms of selenoprotein P. Staining using a digoxigenin-based staining procedure revealed that all five forms contain carbohydrate. This demonstrates that plasma-derived selenoprotein P can be separated into five forms based on SDS-PAGE migration and heparin-Sepharose affinity and that at least two isoforms of selenoprotein P exist based on the presence of 45- and 57-kDa forms. The five forms have been given designations based on their order of elution from the heparin-Sepharose column and their M(r) on SDS-PAGE: selenoprotein P57A (peak 1a, 57 kDa), selenoprotein P45A (peak 1a, 45 kDa), selenoprotein P45B (peak 1b, 45 kDa), selenoprotein P57B (peak 2, 57 kDa), and selenoprotein P57C (peak 3, 57 kDa).