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Title: Characterization of thyroidal glutathione reductase

Abstract

Glutathione levels were determined in bovine and rat thyroid tissue by enzymatic conjugation with 1-chloro-2,4-dinitrobenzene using glutathione S-transferase. Bovine thyroid tissue contained 1.31 {+-} 0.04 mM reduced glutathione (GSH) and 0.14 {+-} 0.02 mM oxidized glutathione (GSSG). In the rat, the concentration of GSH was 2.50 {+-} 0.05 mM while GSSG was 0.21 {+-} 0.03 mM. Glutathione reductase (GR) was purified from bovine thyroid to electrophoretic homogeneity by ion exchange, affinity and molecular exclusion chromatography. A molecular weight range of 102-109 kDa and subunit size of 55 kDa were determined for GR. Thyroidal GR was shown to be a favoprotein with one FAD per subunit. The Michaelis constants of bovine thyroidal GR were determined to be 21.8 {mu}M for NADPH and 58.8 {mu}M for GSSG. The effect of thyroid stimulating hormone (TSH) and thyroxine (T{sub 4}) on in vivo levels of GR and glucose 6-phosphate dehydrogenase were determined in rat thyroid homogenates. Both enzymes were stimulated by TSH treatment and markedly reduced following T{sub 4} treatment. Lysosomal hydrolysis of ({sup 125}I)-labeled and unlabeled thyroglobulin was examined using size exclusion HPLC.

Authors:
Publication Date:
Research Org.:
North Dakota State Univ., Fargo, ND (USA)
OSTI Identifier:
6612843
Resource Type:
Miscellaneous
Resource Relation:
Other Information: Thesis (Ph. D.)
Country of Publication:
United States
Language:
English
Subject:
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.; 59 BASIC BIOLOGICAL SCIENCES; CHLORINATED AROMATIC HYDROCARBONS; METABOLISM; GLUTATHIONE; BIOSYNTHESIS; OXIDOREDUCTASES; CHEMICAL COMPOSITION; CATTLE; CHROMATOGRAPHY; FRACTIONATION; IODINE 125; LIQUID COLUMN CHROMATOGRAPHY; MOLECULAR WEIGHT; NITRO COMPOUNDS; RATS; THYROID; THYROID HORMONES; TRACER TECHNIQUES; ANIMALS; AROMATICS; BETA DECAY RADIOISOTOPES; BODY; DAYS LIVING RADIOISOTOPES; DOMESTIC ANIMALS; DRUGS; ELECTRON CAPTURE RADIOISOTOPES; ENDOCRINE GLANDS; ENZYMES; GLANDS; HALOGENATED AROMATIC HYDROCARBONS; HORMONES; INTERMEDIATE MASS NUCLEI; IODINE ISOTOPES; ISOTOPE APPLICATIONS; ISOTOPES; MAMMALS; NUCLEI; ODD-EVEN NUCLEI; ORGANIC CHLORINE COMPOUNDS; ORGANIC COMPOUNDS; ORGANIC HALOGEN COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; ORGANS; PEPTIDE HORMONES; PEPTIDES; POLYPEPTIDES; PROTEINS; RADIOISOTOPES; RADIOPROTECTIVE SUBSTANCES; RODENTS; RUMINANTS; SEPARATION PROCESSES; SYNTHESIS; VERTEBRATES 560300* -- Chemicals Metabolism & Toxicology; 550201 -- Biochemistry-- Tracer Techniques

Citation Formats

Raasch, R.J. Characterization of thyroidal glutathione reductase. United States: N. p., 1989. Web.
Raasch, R.J. Characterization of thyroidal glutathione reductase. United States.
Raasch, R.J. 1989. "Characterization of thyroidal glutathione reductase". United States. doi:.
@article{osti_6612843,
title = {Characterization of thyroidal glutathione reductase},
author = {Raasch, R.J.},
abstractNote = {Glutathione levels were determined in bovine and rat thyroid tissue by enzymatic conjugation with 1-chloro-2,4-dinitrobenzene using glutathione S-transferase. Bovine thyroid tissue contained 1.31 {+-} 0.04 mM reduced glutathione (GSH) and 0.14 {+-} 0.02 mM oxidized glutathione (GSSG). In the rat, the concentration of GSH was 2.50 {+-} 0.05 mM while GSSG was 0.21 {+-} 0.03 mM. Glutathione reductase (GR) was purified from bovine thyroid to electrophoretic homogeneity by ion exchange, affinity and molecular exclusion chromatography. A molecular weight range of 102-109 kDa and subunit size of 55 kDa were determined for GR. Thyroidal GR was shown to be a favoprotein with one FAD per subunit. The Michaelis constants of bovine thyroidal GR were determined to be 21.8 {mu}M for NADPH and 58.8 {mu}M for GSSG. The effect of thyroid stimulating hormone (TSH) and thyroxine (T{sub 4}) on in vivo levels of GR and glucose 6-phosphate dehydrogenase were determined in rat thyroid homogenates. Both enzymes were stimulated by TSH treatment and markedly reduced following T{sub 4} treatment. Lysosomal hydrolysis of ({sup 125}I)-labeled and unlabeled thyroglobulin was examined using size exclusion HPLC.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = 1989,
month = 1
}

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  • First, the hepatic activity of GSH-T{sub CDNB} was increased only under conditions of severe oxidative stress produced by combined Se- and vitamin E (VE)-deficiency, indicating that VE also affects GSH metabolism. Second, the incorporation of {sup 35}S-methionine into GSH and protein was about 4- and 2-fold higher, respectively, in Se- and VE-deficient chick hepatocytes as compared to controls. Third, chicks injected with the glutathione peroxidase (SeGSHpx) inhibitor, aurothioglucose (AuTG), showed increase hepatic GSH-T{sub CDNB} activity and plasma GSH concentration regardless of their Se status. Fourth, the effect of ascorbic acid (AA), on GSH metabolism was studied. Chicks fed 1000 ppmmore » AA showed decreased hepatic GSH concentration compared to chicks fed no AA in a Se- and VE-deficient diet. Fifth, chicks fed excess Se showed increase hepatic activity of GSH-T{sub CDNB} and GSH concentration regardless of VE status.« less
  • The object of this work was to establish the requirement for GSH and cystine during the activation and proliferation of human peripheral blood mononuclear cells (PBMC). In the author's initial experiments the intracellular GSH content of PBMC was altered by continuous culture or pretreatment with BSO, a specific inhibitor of GSH synthesis. His results demonstrate that, continuous culture of mitogen stimulated PBMC in the presence of BSO inhibited entry into S-phase of the cell cycle and produced a simultaneous decrease in intracellular GSH. The influence of BSO on early activation events were determined by BSO pretreatment. Extensive depletion (>90%) ofmore » the intracellular GSH level prior to mitogenic stimulation did not impair the ability of these cells to produce IL-2 and express IL-2R, indicating that GSH may not be involved in the generation and response to early activation signals. Furthermore, the removal of BSO from these cultures rapidly reversed its inhibitory effects on DNA and GSH synthesis. Cystine transport activities and metabolism by PBMC were characterized in order to examine its contributions to intracellular GSH and early activation proteins. In spite of the ability of cystine to sustain the proliferative response of PBMC, differences in the kinetics of cystine and cysteine uptake indicated that separate transport systems may be operational. Treatment with 2ME enhanced cystine uptake, but lowered the proliferative responses of these cells. Metabolic studies with ({sup 35}S) cystine demonstrated that mitogen stimulation of PBMC enhance cystine uptake.« less
  • Glutathione S-transferases are involved in the biotransformation and/or detoxification of a wide range of organic compounds, including allylic epoxides. GSTs catalyze the transformation of prostaglandin (PG)H[sub 2] into PGE[sub 2] and/or PGF[sub 2alpha]. Specific GST isozymes possessing non-selenium glutathione-peroxidase activity (NonSe-GSH-PX) catalyze the direct reduction of PGH[sub 2] to PGF[sub 2alpha]. Other GST isozymes have been reported to catalyze the transformation of leukotriene (LT)A[sub 4] into LTC[sub 4]. In this study, human liver GSTs were purified and individual isozymes were characterized by SDS electrophoresis, isoelectric focusing, substrate specificities, immunological cross reactivities, and their ability to catalyze the transformation of PGH[submore » 2] to PGF[sub 2alpha], and LTA[sub 4] to LTC[sub 4]. The GST isozyme expression pattern in man varies between individuals. Se-dependent GSH-PX activity (Se-GSH-PX) also plays a role in eicosanoid metabolism. The author infused Se-adequate and Se-deficient dairy cattle with endotoxin into the mammary gland to simulate an inflammation. Arachidonic acid metabolites were extracted and analyzed in both the milk and the milk polymorphonuclear leukocytes (PMNs). PMN's cytosol was assayed for Se- and nonSe-GSH-PX, and GST activity. The results indicate that the Se-deficient cows had lower levels (p < 0.05) of PGE[sub 2] and TXB[sub 2] released into the milk following challenge; however, there was no significant effect on the arachidonic acid metabolites produced by the milk PMNs. Although the Se-deficient cows had significantly lower levels (p < 0.05) of Se-GSH-PX activity, there was no effect on the GST nor NonSe-GSH-PX activity. Overall, eicosanoid biosynthesis is complex, being influenced by both dietary and enzymatic manipulation. The data support Se- and nonSe-GSH-PX playing important roles in eicosanoid formation.« less
  • Biliary excretion, followed by fecal elimination, is a major route for the elimination of metals, including methylmercury; however, the mechanisms for the hepatobiliary transport and metabolism of these compounds have not been identified. The goal of this research was to characterize the mechanism by which methylmercury, a highly neurotoxic environmental contaminant, is transported across the canalicular membrane into bile and to investigate the fate of this metal within the biliary tree. Methylmercury is eliminated predominately through biliary excretion and undergoes extensive enterohepatic cycling. Because methylmercury is believed to be transported across the canalicular membrane into bile as a glutathione complexmore » (CH{sub 3}Hg-SG), its fate may be similar to that of glutathione (GSH). GSH is transported into bile by an electrogenic, carrier mediated transport system, where it is catabolized by the luminal enzymes {gamma}-glutamyltransferase ({gamma}-GT) and dipeptidases yielding glutamate, cysteine, and glycine, and these products are then partially reabsorbed from the biliary tree. To investigate the mechanism by which CH{sub 3}Hg-SG is transported into bile, CH{sub 3}{sup 203}Hg-SG transport was characterized using liver plasma membrane vesicles derived from the canalicular domain of rat hepatocytes. CH{sub 3}Hg-SG uptake was independent of either ATP or a Na{sup +} gradient; however, uptake was stimulated by an induced electrical potential. CH{sub 3}Hg-SG transport was saturable with both high (K{sub m} = 11.9 {+-} 1.6 {mu}M) and low (K{sub m} = 1.47 {+-} 0.22 mM) affinity components. Uptake of this complex was inhibited by GSH and various GSH conjugates but not by GSSG, bile acids, amino acids, or p-glycoprotein substrates. Furthermore, GSH competitively inhibited and trans-stimulated CH{sub 3}Hg-SG uptake.« less
  • The glutathione reductase gene, gor, was cloned from the plant pathogen Xanthomonas campestris pv. phaseoli. Its gene expression and enzyme characteristics were found to be different from those of previously studied homologues. Northern blot hybridization, promoter-lacZ fusion, and enzyme assay experiments revealed that its expression, unlike in Escherichia coli, is OxyR-independent and constitutive upon oxidative stress conditions. The deduced amino acid sequence shows a unique NADPH binding motif where the most highly conserved arginine residue, which is critical for NADPH binding, is replaced by glutamine. Interestingly, a search of the available Gor amino acid sequences from various sources, including othermore » Xanthomonas species, revealed that this replacement is specific to the genus Xanthomonas. Recombinant Gor enzyme was purified and characterized, and was found to have a novel ability to use both, NADPH and NADH, as electron donor. A gor knockout mutant was constructed and shown to have increased expression of the organic peroxide-inducible regulator gene, ohrR.« less