Human Plasma Glutathione Peroxidase Research Articles

The relative effectiveness of human plasma glutathione peroxidase as a catalyst for the reduction of hydroperoxides by glutathione.

To reveal clues to the function of human plasma glutathione peroxidase (GPx), we investigated its catalytic effectiveness with a variety of hydroperoxides. Comparisons of hydroperoxides as substrates for plasma GPx based on the ratio of Vmax/Km were blocked by the limited solubility of the organic hydroperoxides, which prevented kinetic saturation of the enzyme at the chosen glutathione concentration. Therefore, we compared the hydroperoxides by the fold increase in the apparent first-order rate constants of their reactions with glutathione owing to catalysis by plasma GPx. The reductions of aromatic and small hydrophobic hydroperoxides (cumene hydroperoxide, t-amyl hydroperoxide, t-butyl hydroperoxide, paramenthane hydroperoxide) were better catalyzed by plasma GPx than were reductions of the more "physiological" substrates (linoleic acid hydroperoxide, hydrogen peroxide, peroxidized plasma lipids, and oxidized cholesterol).

The crystal structure of seleno-glutathione peroxidase from human plasma at 2.9 Å resolution

Glutathione peroxidase belongs to the family of selenoproteins and plays an important role in the defense mechanisms of mammals, birds and fish against oxidative damage by catalyzing the reduction of a variety of hydroperoxides, using glutathione as the reducing substrate. However, the physiological role of human plasma glutathione peroxidase remains unclear due to the low levels of reduced glutathione in human plasma and the low reactivity of this enzyme. The crystal structure of human plasma glutathione peroxidase was determined by Patterson search methods using a polyalanine model modified from the known structure of bovine erythrocyte glutathione peroxidase. The structure was refined to a crystallographic R-factor of 0.228 ( R free = 0.335) with I > 2σ( I) reflections in the resolution range of 8 to 2.9 Å. The asymmetric unit contains a dimer. Tetramers are built up from dimers by crystallographic symmetry. The subunit structure of the plasma enzyme shows the typical structure motif of the thioredoxin fold consisting of a central β-sheet and several flanking α-helices. The active site selenocysteine residue is situated in the loop between β1 and α1 and is located in a pocket on the protein surface. The overall structure of the human plasma enzyme is similar to that of the bovine erythrocyte enzyme. The main differences in their subunit structures are an extended N terminus and the possible existence of a disulfide bridge in the plasma enzyme. Compared to the bovine erythrocyte enzyme, a number of residues in the active site are mutated or deleted in the plasma enzyme, including all the residues that were previously suggested to be involved in glutathione binding. The observed structural differences between the two enzymes suggest differences in substrate binding and specificity.

The thioredoxin and glutaredoxin systems are efficient electron donors to human plasma glutathione peroxidase.

Human plasma glutathione peroxidase (GSH-Px) is a distinct extracellular selenoenzyme that detoxifies hydroperoxides when used with GSH in high (mM) non-physiological concentrations. We have discovered that NADPH and human thioredoxin reductase (TR) by itself or with thioredoxin (Trx) are efficient electron donors to this human plasma peroxidase. Incubation of 0.05 microM TR with 0.25 microM GSH-Px, in a system free from GSH, resulted in reduction of t-butyl hydroperoxide. Addition of Trx, 2.5 and 5 microM, respectively, further increased the rate of the reaction. These data were obtained using an assay measuring the oxidation of NADPH. A direct assay demonstrated the formation of cumyl alcohol from cumene hydroperoxide in this GSH-independent peroxidase reaction. Incubation of 0.25 microM GSH-Px with a low concentration of GSH (10 microM), representing the upper level in plasma, plus excess glutathione reductase and NADPH did not result in any reduction of t-butyl hydroperoxide. However, after addition of 2.5 microM human glutaredoxin, a linear peroxidase reaction started. The results suggest that extracellular TR, Trx, or glutaredoxin are reductants for the selenium-dependent peroxidase rather than GSH.

Mouse plasma glutathione peroxidase. cDNA sequence analysis and renal proximal tubular expression and secretion.

A mouse kidney cDNA isolated by differential screening was found to be highly homologous to rat, human, and bovine plasma glutathione peroxidase (GPx) sequences. Analysis of the full-length coding region sequence demonstrated an in-frame selenocysteine-encoding opal codon and putative signal sequence, suggesting that the sequence represents the mouse homolog of plasma GPx. The level of expression of plasma GPx in various mouse tissues and during development was investigated by Northern blot analysis. Plasma GPx mRNA was observed to be very abundant in kidney compared with placenta, epididymis, intestine, lung, heart, testis, ovary, salivary gland, spleen, thymus, stomach, brain, and fetal kidney and could not be detected in pancreas or in liver except from pregnant mice. In addition, plasma GPx mRNA levels were shown to increase during postnatal development of the kidney. In situ hybridization localized plasma GPx mRNA to proximal tubules, while primary cell culture demonstrated that plasma GPx is synthesized and secreted by proximal tubular epithelial cells. The relative abundance of plasma GPx mRNA in mouse kidney suggests that proximal tubules may be the primary source of the enzyme detectable in plasma and further suggests that plasma GPx has an important function in protecting the kidney from oxidative damage.

The human plasma glutathione peroxidase-encoding gene: organization, sequence and localization to chromosome 5q32

A genomic clone encoding the human plasma glutathione peroxidase (PGPx), a major enzyme in reducing lipid hydroperoxide and hydrogen peroxide in plasma, was isolated and 5618 nucleotides (nt) were determined. The nt sequence data revealed that the PGPx gene is composed of five exons spanning approx. 10 kb. Primer extension experiments mapped the transcription start point at 298 nt upstream from the predicted start codon. Twenty nt upstream from the polyadenylation site of the gene, an uncanonical polyadenylation signal, AGTAAA, was found. Human PGPx was localized on chromosome 5 band q32 by fluorescence in situ hybridization.

Expression, characterization, and tissue distribution of a new cellular selenium-dependent glutathione peroxidase, GSHPx-GI.

We have characterized a new selenium-dependent glutathione peroxidase, GSHPx-GI, by expressing a GSHPx-GI cDNA isolated from human hepatoma HepG2 cells in human mammary carcinoma MCF-7 cells, which have virtually undetectable expression of either the classical cellular enzyme, GSHPx-1, or GSHPx-GI at the protein level. One of the G418-resistant clones, neo-D1, expresses the transfected GSHPx-GI cDNA. This is based on 1) the presence of an additional GSHPx-GI DNA restriction fragment detected by Southern analysis; 2) the presence of a 1.9-kilobase (kb) GSHPx-GI mRNA in addition to the 1.0-kb endogenous mRNA by Northern analysis; and 3) the appearance of a 22-kDa 75Se-labeled protein which is absent in parental MCF-7 cells revealed by SDS-polyacrylamide gel electrophoresis. GSHPx-GI expressed in neo-D1 is a tetrameric protein localized in cytosol. GSHPx-GI does not cross-react with antisera against human GSHPx-1 or human plasma glutathione peroxidase (GSHPx-P). Similar substrate specificities are found for GSHPx-1 and GSHPx-GI; they both catalyze the reduction of H2O2, tert-butyl hydroperoxide, cumene hydroperoxide, and linoleic acid hydroperoxide with glutathione, but not of phosphatidylcholine hydroperoxide. GSHPx-GI mRNA was readily detected in human liver and colon, and occasionally in human breast samples, but not other human tissues including kidney, heart, lung, placenta, or uterus. In rodent tissues, GSHPx-GI mRNA is only detected in the gastrointestinal tract, and not in other tissues including liver. In fact, GSHPx-GI appears to be the major glutathione-dependent peroxidase activity in rodent GI tract. This finding suggests that GSHPx-GI could play a major role in protecting mammals from the toxicity of ingested lipid hydroperoxides. In conclusion, we have demonstrated that GSHPx-GI is the fourth member in the selenium-dependent glutathione peroxidase family, in addition to GSHPx-1, GSHPx-P, and phospholipid hydroperoxide glutathione peroxidase (PHGPX).

ChemInform Abstract: Primary Structure of Human Plasma Glutathione Peroxidase Deduced from cDNA Sequences

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Detection of a higher oxidation state of manganese-prostaglandin endoperoxide synthase.

Addition of arachidonic acid or 5-phenyl-4-pentenylhydroperoxide to manganese-prostaglandin endoperoxide synthase (Mn-PGH synthase) produced a species with an absorbance maximum at 418 nm. This maximum is distinct from those of resting enzyme (372 and 468 nm) or reduced enzyme (434 nm). The formation of the 418 nm-absorbing species was observed immediately after the addition of hydroperoxide to enzyme but only after a 10-s lag period following addition of arachidonate. Mn-PGH synthase exhibited a peroxidase activity that was 0.8% that of Fe-PGH synthase. Addition of peroxidase reducing substrates to the oxidized form of Mn-PGH synthase diminished the absorbance at 418 nm. In the case of N,N,N',N'-tetramethylphenylenediamine, reduction of the 418 nm-absorbing species was accompanied by an increase in absorbance at 610 nm due to the oxidized form of the amine. Thus, the spectral and chemical properties of the 418 nm-absorbing species are consistent with its existence as a higher oxidation state of Mn-PGH synthase. Kinetic analysis indicated that formation of the higher oxidation state preceded or was coincident with oxygenation of the fatty acid substrate, eicosa-11,14-dienoic acid. The cyclooxygenase activity of Mn-PGH synthase was inhibited by the combination of glutathione and human plasma glutathione peroxidase at a glutathione peroxidase concentration 227-fold lower than the concentration that inhibited Fe-PGH synthase. The results suggest that Mn-PGH synthase forms a higher oxidation state following reaction with hydroperoxides added exogenously or generated endogenously from polyunsaturated fatty acid substrates. This higher oxidation state functions in the peroxidase catalytic cycle of Mn-PGH synthase, and its formation appears to be essential for activation of the cyclooxygenase catalytic cycle.

Primary Structure of Human Plasma Glutathione Peroxidase Deduced From cDNA Sequences

Abstract Human plasma glutathione peroxidase (GSHPx) has been shown to be a selenium-containing enzyme immunologically distinct from cellular GSHPx. Oligonucleotide probes, based on a partial amino acid sequence of one peptide in a lysine endopeptidase-digest of the purified enzyme, were used to screen a human placenta cDNA library. Nucleotide sequence analysis of the obtained clones revealed that GSHPx consisted of a 678-base pair open reading frame coding for a 226-amino acid polypeptide. The in-frame TGA codon observed at positions 217–219 was assigned to selenocysteine. The amino acid sequence exhibited only 44% homology with that of human cellular GSHPx. Northern blot analysis revealed a single transcript of 2.2 kilobases in the poly (A)+ RNA fractions of human placenta and HepG2 (human hepatic cell line), but not that of human liver. The transcript was also detected in rat kidney, but not in rat liver, lung, heart and brain.

Partial Sequence of Human Plasma Glutathione Peroxidase and Immunologic Identification of Milk Glutathione Peroxidase as the Plasma Enzyme

Plasma glutathione peroxidase (p-GSHPx) is a unique selenoglycoprotein. A hepatic cell line synthesizes both this extracellular form for secretion and the cellular form that remains within the cells. Because the two forms could be a result of post-translational modifications of a product of a single gene, we partially sequenced p-GSHPx. Purified p-GSHPx was trypsin digested, and three of the peptides were sequenced. Only one of the peptide sequences was partially homologous to a sequence found in human cellular glutathione peroxidase. Because p-GSHPx is a secreted enzyme, we determined whether GSHPx in milk (another extracellular fluid) is due to this form of the enzyme. Ninety percent of human milk GSHPx activity could be precipitated by anti-p-GSHPx-immunoglobulin G. Thus, most, if not all, GSHPx activity in milk is due to the plasma selenoprotein form of the enzyme. In milk of two North American women, 3.6% and 14.3% of selenium was associated with GSHPx.

Characterization and partial amino acid sequence of human plasma glutathione peroxidase

Human plasma glutathione peroxidase was purified to homogeneity and partially sequenced. Overlapping peptide fragments from three endopeptidase digests permitted the determination of one sequence of 32 contiguous amino acids and one sequence of 23 contiguous amino acids. Five additional unique peptide sequences without obvious overlaps were obtained. The sequence of 32 amino acid residues aligns with positions 82–113 of human cytosolic glutathione peroxidase with nine mismatches without gaps or insertions. The sequence of 23 amino acid residues aligns with positions 157–178 with six mismatches and an insertion of one residue. Three additional peptide sequences with no obvious sequence homology to glutathione peroxidase can be aligned based on the sequence of a cDNA clone encoding plasma glutathione peroxidase that was isolated from a human placental library. The plasma enzyme is a homotetramer composed of 21-kDa subunits which cannot reduce phospholipid hydroperoxides. These results indicate that the plasma glutathione peroxidase is distinct from both the classical cytosolic enzyme and the monomeric phospholipid hydroperoxide glutathione peroxidase. Only a negligible amount of glutathione peroxidase activity was detected in bile, indicating that the liver exports plasma glutathione peroxidase exclusively to the circulation.

Primary Structure of Human Plasma Glutathione Peroxidase Deduced from eDNA Sequences1

Human plasma glutathione peroxidase (GSHPx) has been shown to be a selenium-containing enzyme immunologically distinct from cellular GSHPx. Oligonucleotide probes, based on the partial amino acid sequence of plasma GSHPx, were synthesized and used to screen a human placenta cDNA library. Nucleotide sequence analysis of the obtained clones revealed that GSHPx consisted of a 678-base pair open reading frame coding for a 226-amino acid polypeptide with a Mr of 25,389. About 50% of the deduced amino acid sequence was confirmed by partial amino acid sequencing of the peptides in a lysine endopeptidase-digest of the purified enzyme. The amino acid sequence exhibited only 44% homology with that of human cellular GSHPx. Northern blot analysis revealed a single transcript of 2.2 kilobases in the poly(A)+ RNA fractions of human placenta and HepG2 (a human hepatic cell line), but not that of human liver and endothelial cells.

Human plasma glutathione peroxidase is a distinct protein from intracellular glutathione peroxidase

Human plasma glutathione peroxidase is a distinct protein from intracellular glutathione peroxidase

Plasma Selenium-dependent Glutathione Peroxidase: Cell of origin and secretion

Human plasma glutathione peroxidase (GSHPx) has been shown to be a glycosylated selenoprotein distinct enzymatically, structurally, and antigenically from known cellular glutathione peroxidases. The extracellular location of the enzyme and the fact that it is glycosylated suggested that it is a secreted protein. Utilizing mutually non-cross-reactive antibodies to human cellular and plasma GSHPx, we conducted a search to determine the tissue of origin for plasma GSHPx. The cells screened were endothelial cells because they are the main source of extracellular superoxide dismutase, HL-60 cells (myeloid cell line) because they are the main source of extracellular H2O2, and Hep G2 cells (hepatic cell line) because they are the source of many plasma proteins. Human umbilical vein endothelial cells were metabolically labeled with either [35S]methionine or [75Se]selenious acid, and HL-60 cells and Hep G2 cells were metabolically labeled with [75Se]selenious acid. Proteins were immunopurified from the labeled cells and their media with either anti-red blood cell (RBC) GSHPx IgG or with anti-plasma GSHPx IgG. Utilizing anti-RBC GSHPx IgG, only the cellular form of the enzyme was precipitated from all the cells tested but not from their media. When anti-plasma GSHPx IgG was applied to the cells and their media, a selenoprotein was precipitated only from the media of Hep G2 cells. When Hep G2 cells were incubated in the presence of the carboxylic ionophore monensin, an intracellular selenoprotein could be detected using anti-plasma GSHPx IgG. The precipitation of the cellular form from all three cell types was partially inhibited by preincubation of the anti-RBC GSHPx IgG with purified RBC GSHPx while the precipitation of the selenoprotein from the medium of Hep G2 cells by anti-plasma GSHPx IgG was prevented by preincubation of the antibody with purified plasma GSHPx. We suggest that plasma GSHPx is synthesized by and secreted from hepatic cells. This is, to the best of our knowledge, the only known selenoprotein with a defined function that has been shown to be synthesized for secretion by mammalian cells.

Characterization of the major hydroperoxide-reducing activity of human plasma. Purification and properties of a selenium-dependent glutathione peroxidase.

We have recently characterized the major hydroperoxide-reducing enzyme of human plasma as a glutathione peroxidase (Maddipati, K. R., Gasparski, C., and Marnett, L. J. (1987) Arch. Biochem. Biophys. 254, 9-17). We now report the purification and kinetic characterization of this enzyme. The purification steps involved ammonium sulfate precipitation, hydrophobic interaction chromatography on phenyl-Sepharose, anion exchange chromatography, and gel filtration. The purified peroxidase has a specific activity of 26-29 mumol/min/mg with hydrogen peroxide as substrate. The human plasma glutathione peroxidase is a tetramer of identical subunits of 21.5 kDa molecular mass as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and is different from human erythrocyte glutathione peroxidase. The plasma peroxidase is a selenoprotein containing one selenium per subunit. Unlike several other glutathione peroxidases this enzyme exhibits saturation kinetics with respect to glutathione (Km for glutathione = 4.3 mM). The peroxidase exhibits high affinity for hydroperoxides with Km values ranging from 2.3 microM for 13-hydroperoxy-9,11-octadecadienoic acid to 13.3 microM for hydrogen peroxide at saturating glutathione concentration. These kinetic parameters are suggestive of the potential of human plasma glutathione peroxidase as an important regulator of plasma hydroperoxide levels.

Properties of glutathione peroxidase isolated from human plasma

Human plasma glutathione peroxidase (GPx) was purified to homogeneity by ammonium sulfate fractionation, gel filtration on Sephadex G-150, chromatography on DEAE Sephacel, chromatofocusing with polybuffer, and gel filtration with Sephadex G-75. This isolation resulted in about 5,400-fold purification of the enzyme with a 32% yield in enzyme activity. The final preparation had a specific activity of about 28 units (mmoles NADPH oxidized) per milligram of protein. Determination of selenium on the purified enzyme revealed a content of 3.8 g atoms per mole GPx. Gel electrophoresis using SDS with standard proteins revealed a molecular weight of about 23,000 for the subunits, which would indicate a molecular weight of about 92,000 for the native enzyme. Amino acid analyses of the purified GPx indicated aspartate, glutamate, proline, glycine, alanine, and leucine as the predominant amino acids and cysteine, methionine, tryptophan, and histidine as the minor amino acids.

Purification and characterization of human plasma glutathione peroxidase: A selenoglycoprotein distinct from the known cellular enzyme

Glutathione peroxidase (GSHPx), (glutathione:H 2O 2 oxidoreductase, EC 1.11.1.9) was purified to homogeneity from human plasma. This resulted in a 6800-fold purification of the enzyme with a 2.8% yield. The purification process involved ammonium sulfate fractionation, DEAE-cellulose batch and column chromatographies, hydroxyapatite, and Sephadex G-200 and DEAE-Sephadex A-25 chromatographies. The major peak on DEAE-Sephadex A-25 column chromatography was found to be homogeneous on poly-acrylamide gel electrophoresis in the presence or absence of sodium dodecyl sulfate (SDS). Relative mobility in nondenaturing polyacrylamide gel electrophoresis at pH 8.2 was 0.5 for the purified enzyme as detected by both protein staining and enzyme activity compared with 0.38 for erythrocyte GSHPx. The molecular weight of the plasma enzyme as determined by gel filtration was found to be approximately 100,000. SDS-gel electrophoresis of the plasma enzyme gave a subunit molecular weight of approximately 23,000. This suggests that the plasma enzyme exists as a tetramer in its native state, similar to that seen for the erythrocyte enzyme, but with slightly different mobility on SDS-gel electrophoresis. Plasma GSHPx, like the erythrocyte enzyme, was found to contain approximately four atoms of selenium per mole of protein. Utilizing iodinated concanavalin A, it was found that plasma GSHPx, but not the erythrocyte GSPx, is a glycoprotein. Purified plasma enzyme catalyzes both the reduction of tertiary butyl hydroperoxide and hydrogen peroxide. The apparent K m of plasma GSHPx for GSH is 5.3 m m and for tertiary butyl hydroperoxide it is 0.57 m m. Copper, mercury, and zinc strongly inhibit the enzyme activity of plasma GSHPx. Rabbit antibodies directed against the human erythrocyte GSHPx do not precipitate the enzyme activity of the purified plasma enzyme. Radioimmunoassay utilizing erythrocyte GSHPx and anti-erythrocyte GSHPx antibodies showed that less than 0.13% of the antigenically detectable protein is found in the purified GSHPx from plasma.