Purification and characterization of rat brain cytosolic 3,5,3'-triiodo-L-thyronine-binding protein. Evidence for binding activity dependent on NADPH, NADP and thioredoxin.

A rat brain cytosolic 3,5,3'-triiodo-L-thyronine-(T3)-binding protein (CTBP) was purified using, successively, carboxymethyl-Sephadex, DEAE-Spherodex, T3-Sepharose-4B affinity chromatography and Sephacryl S-200. The molecular mass determined by SDS/PAGE wa 58 kDa. The binding characteristics determined by Scatchard analysis revealed a single class of binding sites with a Ka of 1.56 nM-1 and a maximal binding capacity of 7500 nmol T3/g protein. The relative binding affinities of iodothyronine analogues were D-T3 > L-T3 > L-T4 > 3,3'-5-triiodothyroacetic acid > reverse T3. The optimum pH for binding was 7.5. Purified brain CTBP was reversibly inactivated by charcoal. NADPH, NADP and thioredoxin restored binding activity to a level higher than that of the control; this effect was concentration dependent. Maximal activation was observed at 25 nM NADPH. NADP was effective only in the presence of 1 mM dithiothreitol; maximal activity was obtained at 10 nM NADP. At concentrations higher than 50 nM NADP, the binding gradually decreased. Thioredoxin in the presence of 1 mM dithiothreitol activated CTBP; maximal binding was obtained with 4 microM thioredoxin. In the presence of NADPH, NADP or thioredoxin the maximal binding capacity increased 2-4 times and the Ka was 2.6 nM-1. These results show that the activity of purified cytosolic brain T3-binding protein may be modulated by NADPH, NADP or thioredoxin.     

Activation of translation via reduction by thioredoxin-thioredoxin reductase in Saccharomyces cerevisiae

Previously we reported that in vitro translation activity in extracts of Saccharomyces cerevisiae was stimulated by dithiothreitol (DTT) and further increased by the addition of thioredoxin (TRX1) [Choi, S.K. (2007) Thioredoxin-mediated regulation of protein synthesis by redox in Saccharomyces cerevisiae. Kor. J. Microbiol. Biotechnol. 35, 36-40]. To identify the pathway affecting translation, we cloned and purified thioredoxin reductase 1 (TRR1), thioredoxin reductase 2 (TRR2), glutaredoxin 1 (GRX1) and glutaredoxin reductase 1 (GLR1) as fusion proteins. Thioredoxin-mediated activation of translation was more effectively stimulated by NADPH or NADH than by DTT. Moreover, addition of TRR1 led to a further increase of translation in the presence of thioredoxin plus NADPH. These findings indicate that redox control via the thioredoxin-thioredoxin reductase system plays an important role in the regulation of translation.     

Thionamides and arsenite inhibit specific T3 binding to the hepatic nuclear receptor

Methimazole (MMI) and propylthiouracil (PTU) are widely used for the treatment of Graves' disease. However, no studies have been reported on the action of these drugs on binding of L-triiodothyronine (T3) to the nuclear receptor. T3 receptors of rat liver nuclei, prepared by differential centrifugation, were extracted with 0.4 M KCl and 5 mM dithiothreitol (DTT). In the assessment of T3 binding to the DTT-reduced receptor, the hepatic nuclear extract was chromatographed on Superose 6 to remove DTT and isolate proteins of relative mass approximately 50,000 (chromatographed nuclear receptors (CNRs)), prior to the addition of [125I]T3 of high specific activity (3300 microCi/micrograms; 1 Ci = 37 GBq). MMI or PTU at 2 mM reduced specific T3 binding to CNR by 84% and 85%, respectively. The inhibitory effects of these reagents and 2 mM sodium arsenite (which complexes dithiols) were additive. Scatchard analyses indicated that neither MMI nor PTU (at 2 mM) significantly altered the affinity constant (Ka) (from 2.41 x 10(9) to 1.74 x 10(9) M-1 for PTU and 1.79 x 10(9) M-1 for MMI), while they both decreased (p less than 0.02) maximal binding capacity (from 0.36 +/- 0.02 to 0.19 +/- 0.02 pmol/mg protein for MMI and 0.17 +/- 0.02 pmol/mg protein for PTU). Dose-response curves showed that 50% inhibition was attained at 0.6 mM PTU or 1.0 mM MMI with approximately 25% inhibition by both at 0.1 mM. Artefactual binding effects by MMI and PTU on [125I]T3 were excluded by chromatography experiments. Similar results were obtained using nuclear receptors prepared from livers of hyperthyroid rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of adrenaline pretreatment on in vitro binding of 125I-triiodothyronine to nuclear receptor, intracellular distribution of endogenous triiodothyronine and activities of alfa-glycerophosphate dehydrogenase and malic enzyme in rat liver

Especially coated adrenaline tablets (A) or placebo tablets (P) which release linearly the hormone were implanted in male Wistar rats. Six hours later animals were sacrificed and kinetic parameters of T3-125I binding to nuclear receptor, intracellular distribution of endogenous T3 and activities of alfa-GPD and ME were investigated. The association constant values (Ka) of nuclear receptor were increased after pretreatment with 7.5, 15 and 45 mg A tablets and were 1.07, 1.35 and 1.48 X 10(9) M-1 vs 0.85 X 10(8) M-1 value seen after P. The maximal binding capacity (MBC) values decreased after pretreatment with the same doses of A and were 0.044, 0.036 and 0.025 pmol T3/100 micrograms DNA vs. 0.065 pmol T3/100 micrograms DNA in P pretreated. Adrenaline pretreatment significantly increased the amount of endogenous T3 present in liver nuclei while the amount of T3 present in cytosol decreased. Activity of mitochondrial alfa-GPD was increased after 15 and 45 mg of A. Significant rise of activity of cytosol ME was seen only after pretreatment with 45 mg of A.     

Thioredoxin/Glutaredoxin System of Chlorella: CHLORELLA ADENOSINE 5'-PHOSPHOSULFATE SULFOTRANSFERASE CANNOT USE THIOREDOXIN OR GLUTAREDOXIN AS COFACTORS

Using the thioredoxin/glutaredoxin-dependent adenosine 3'-phosphate 5'-phosphosulfate reductase coupled assay system, the Chlorella thioredoxin/glutaredoxin system has been partially purified and characterized. A NADPH-thioredoxin reductase and two thioredoxin/glutaredoxin activities, designated as Chlorella thioredoxin/glutaredoxin protein I and II (CPI and CPII), were found in crude extracts of Chlorella. Similar to their counterparts from Escherichia coli, both CPI and CPII are heat-stable low molecular proteins of approximately 14,000. While CPI (but not CPII) is a substrate for its homologous NADPH-thioredoxin reductase as well as for E. coli NADPH-thioredoxin reductase, CPII is better than CPI as a substrate for reduction by the glutathione system. Based on these properties, CPI and CPII may be classified as Chlorella thioredoxin and Chlorella glutaredoxin, respectively. The Chlorella NADPH-thioredoxin reductase (M(r) = 72,000, with two 36,000-dalton subunits) resembles E. coli-thioredoxin reductase in size. Besides Chlorella thioredoxin, the Chlorella thioredoxin reductase will also use E. coli thioredoxin, but not glutaredoxin, as a substrate. Although a thioredoxin-like protein has been implicated in higher plant light-dependent sulfate reaction, neither Chlorella thioredoxin nor glutaredoxin can stimulate the thiol-dependent adenosine 5'-phosphosulfate-sulfotransferase reaction. Furthermore, Chlorella thioredoxin and glutaredoxin, in conjunction with an appropriate reductase system, cannot replace the thiol requirement of Chlorella adenosine 5'-phosphosulfate-sulfotransferase. The exact physiological roles and subcellular localization of the Chlorella thioredoxin and glutaredoxin systems remain to be determined.     

Thioredoxin and related proteins in procaryotes

Thioredoxin is a small (Mr 12,000) ubiquitous redox protein with the conserved active site structure: -Trp-Cys-Gly-Pro-Cys-. The oxidized form (Trx-S2) contains a disulfide bridge which is reduced by NADPH and thioredoxin reductase; the reduced form [Trx(SH)2] is a powerful protein disulfide oxidoreductase. Thioredoxins have been characterized in a wide variety of prokaryotic cells, and generally show about 50% amino acid homology to Escherichia coli thioredoxin with a known three-dimensional structure. In vitro Trx-(SH)2 serves as a hydrogen donor for ribonucleotide reductase, an essential enzyme in DNA synthesis, and for enzymes reducing sulfate or methionine sulfoxide. E. coli Trx-(SH)2 is essential for phage T7 DNA replication as a subunit of T7 DNA polymerase and also for assembly of the filamentous phages f1 and M13 perhaps through its localization at the cellular plasma membrane. Some photosynthetic organisms reduce Trx-S2 by light and ferredoxin; Trx-(SH)2 is used as a disulfide reductase to regulate the activity of enzymes by thiol redox control. Thioredoxin-negative mutants (trxA) of E. coli are viable making the precise cellular physiological functions of thioredoxin unknown. Another small E. coli protein, glutaredoxin, enables GSH to be hydrogen donor for ribonucleotide reductase or PAPS reductase. Further experiments with molecular genetic techniques are required to define the relative roles of the thioredoxin and glutaredoxin systems in intracellular redox reactions.     

Thioredoxin and related proteins in procaryotes

Abstract Thioredoxin is a small ( M _r 12,000) ubiquitous redox protein with the conserved active site structure: -Trp-Cys-Gly-Pro-Cys-. The oxidized form (Trx-S₂) contains a disulfide bridge which is reduced by NADPH and thioredoxin reductase; the reduced form [Trx(SH)₂] is a powerful protein disulfide oxidoreductase. Thioredoxins have been characterized in a wide variety of prokaryotic cells, and generally show about 50% amino acid homology to Escherichia coli thioredoxin with a known three-dimensional structure. In vitro Trx-(SH)₂ serves as a hydrogen donor for ribonucleotide reductase, an essential enzyme in DNA synthesis, and for enzymes reducing sulfate or methionine sulfoxide. E. coli Trx-(SH)₂ is essential for phage T7 DNA replication as a subunit of T7 DNA polymerase and also for assembly of the filamentous phages f1 and M13 perhaps through its localization at the cellular plasma membrane. Some photosynthetic organisms reduce Trx-S₂ by light and ferrodoxin; Trx-(SH)₂ is used as a disulfide reductase to regulate the activity of enzymes by thiol redox control.
Thioredoxin-negative mutants ( trxA ) of E. coli are viable making the precise cellular physiological functions of thioredoxin unknown. Another small E. coli protein, glutaredoxin, enables GSH to be hydrogen donor for ribonucleotide reductase or PAPS reductase. Further experiments with molecular genetic techniques are required to define the relative roles of the thioredoxin and glutaredoxin systems in intracellular redox reactions.     

Evidence for the presence of two active forms of cytosolic 3,5,3'-triiodo-L-thyronine (T3)-binding protein (CTBP) in rat kidney. Specialized functions of two CTBPs in intracellular T3 translocation

Cytosolic NADPH-dependent 3,5,3'-triiodo-L-thyronine (T3)-binding protein (CTBP) purified from rat kidney was further characterized in its T3 binding and its interaction with nuclei. Pretreatment of the CTBP with NADP induced dithiothreitol (DTT)-dependent T3 binding. The DTT-dependent T3 binding was increased by NADP in a concentration-dependent manner, and the maximal binding was obtained by 0.1 microM NADP. Higher concentrations of NADP (more than 0.1 microM), however, reduced T3 binding. NAD also induced DTT-dependent T3 binding, but was very low compared to that induced by NADP. NADPH and NADH did not produce DTT-dependent T3 binding. This NADP-activated, DTT-dependent T3 binding was characterized as follows: Ka for T3 binding was 1.8 x 10(9) M-1, and the maximal binding capacity was 15,000 pmol/mg of protein in the CTBP activated by 0.1 microM NADP. The molecular weight of the CTBP was 58,000 (4.7 S). A complex of [125I]T3 and CTBP (NADP.DTT.CTBP.[125I]T3), which was made from the CTBP pretreated with NADP and DTT, did not bind to DNA. However, the complex bound to the nuclei prepared from rat kidney. Treatment of the nuclei with 0.38 M KCl and with DNase I did not lead to loss of the binding activity for the complex. Treatment of nuclei with 0.5 M NaCl led to the loss of the activity for binding the complex. A complex of [125I]T3 and NADPH-activated CTBP did not bind these nuclear preparations. These results suggested that the active form of CTBP is present in two different forms: one is NADPH-activated, which plays a role as a reservoir for cytoplasmic T3, and the other is NADP-activated, which plays a role as a T3 carrier protein that transfers T3 from cytoplasm to nucleus.     

Thyroid hormone receptors in brain and liver during ageing

The binding properties--binding capacity (MBC) and affinity (Ka)--of T3 nuclear receptors were analyzed in cortex, cerebellum and liver of rats aged 3, 6, 12 and 24 months. A slight but not significant decrease of Ka was observed in different tissues of normal rats. In hypothyroid animals the Ka in cortex at 24 months was significantly lower than at 3 months. During ageing the MBC of brain receptors decreased whereas hepatic receptors were not altered. Hypothyroidism did not further affect the MBC of the receptors. The data indicate that during ageing the T3 nuclear receptors behave differently in brain and liver. The difference in MBC suggests selectivity in organ sensitivity to thyroid hormones.     

Analysis of nuclear 3,3',5-triiodothyronine receptor in the brown adipose tissue (BAT) of the postnatal lamb.

Postnatal thermogenesis in sheep is associated with increased sympathoadrenal activities, a T3 surge and an enhanced brown adipose tissue (BAT) type II 5'-monodeiodinating (5'-MDI) activity. The latter peaks 3-4 days after birth and is known to be important in generating intracellular T3 for nuclear receptor binding. In order to further investigate the mechanism(s) responsible for neonatal thermogenesis, thyroid hormone nuclear receptor (T3NR) binding characteristics were quantified in lamb BAT from newborn (NB) to 30d of postnatal age. Maximal binding capacities (MBC, mean +/- SEM fmoles T3/mg DNA) in BAT showed a decrease as studied by ANOVA during the first 11 days (NB to 1d, 148 +/- 24 [N = 5, p < 0.01, cf. 3-5d group]; 3-5 d, 61 +/- 5.5 [N = 5]; 10-11d, 72 +/- 9.1 [N = 4]). Afterwards, MBC increased at 30d (196 +/- 32, N = 4, p less than 0.01, cf. 3-5d group). BAT T3NR binding affinities (10(9) M-1) were comparable in all age groups studied (NB-1d, 2.8 +/- 0.3; 3-5d, 3.4 +/- 0.3; 10-11d, 4.0 +/- 1.1; 30d, 2.4 +/- 0.4). The data suggest that the postnatal surge in T3 and type II 5'-MDI is accompanied with a concurrent decrease in MBC of BAT T3NR. The latter may represent a down-regulation of T3NR presumably in an attempt to regulate the overall effect of thyroid hormone in neonatal thermogenesis.     

Proof that the endogenous, heat-stable glucocorticoid receptor-activating factor is thioredoxin

Extraction of rat liver cytosol with charcoal inactivates glucocorticoid-binding capacity and receptors can be reactivated to the steroid-binding state by an endogenous reducing system utilizing NADPH and a Mr = 12,000, heat-stable, endogenous, cytosolic protein (Grippo, J. F., Tienrungroj, W., Dahmer, M. K., Housley, P. R., and Pratt, W. B. (1983) J. Biol. Chem. 258, 13658-13664). In this paper we show that NADPH-dependent conversion of the rat liver glucocorticoid receptor from a nonbinding to a steroid-binding form is blocked in an immune-specific manner by antisera raised against purified rat liver thioredoxin reductase or thioredoxin. The inhibition produced by thioredoxin reductase antiserum may be circumvented by dithiothreitol or overcome by addition of purified thioredoxin reductase. These observations prove that the endogenous glucocorticoid receptor-activating factor is thioredoxin and that the enzyme required for generating the steroid-binding conformation of the glucocorticoid receptor by the endogenous receptor-activating system is thioredoxin reductase.     

Alternative mRNAs arising from trans-splicing code for mitochondrial and cytosolic variants of Echinococcus granulosus thioredoxin Glutathione reductase

Thioredoxin and glutathione systems are the major thiol-dependent redox systems in animal cells. They transfer via the reversible oxidoreduction of thiols the reducing equivalents of NADPH to numerous substrates and substrate reductases and constitute major defenses against oxidative stress. In this study, we cloned from the helminth parasite Echinococcus granulosus two trans-spliced mRNA variants that encode thioredoxin glutathione reductases (TGR). These variants code for mitochondrial and cytosolic selenocysteine-containing isoforms that possess identical glutaredoxin (Grx) and thioredoxin reductase (TR) domains and differ exclusively in their N termini. Western blot analysis of subcellular fractions with specific anti-TGR antibodies showed that TGR is present in both compartments. The biochemical characterization of the native purified TGR suggests that the Grx and TR domains of the enzyme can function either coupled or independently of each other, because the Grx domain can accept electrons from either TR domains or the glutathione system and the TR domains can transfer electrons to either the fused Grx domain or to E. granulosus thioredoxin.     

Reduction of castor-seed 2S albumin protein by thioredoxin

The 2S albumin from the endosperm of castor seed (Ricinus communis L.) seed was reduced by thioredoxin from either wheat germ or Escherichia coli. The 2S protein is made up of a large (approx. 7 kDa) subunit that contains two intramolecular disulfides and a small (approx. 4 kDa) subunit that lacks intramolecular disulfides. The two subunits are joined by at least one intermolecular disulfide bond. Thioredoxin could be reduced either enzymically with NADPH and NADP-thioredoxin reductase or chemically with dithiothreitol. Reduced glutathione and glutaredoxin (from E. coli) were without effect. The ability of the 2S protein to undergo reduction by thioredoxin was demonstrated by a direct reduction procedure based on the fluorescent probe, monobromobimane, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and by an enzymatic procedure in which reduction is linked to activation of chloroplast NADP-malate dehydrogenase. Analyses indicated that thioredoxin actively reduced the intramolecular disulfides of the 2S large subunit, but was ineffective in reducing the intermolecular disulfide(s) that connect the large to the small subunit. These findings extend the role of thioredoxin to the reduction of a seed protein that is widely distributed in oil producing plants.Abbreviations DDT dithiothreitol - mBBr monobromobimane - NTR NADP-thioredoxin reductase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis This work was supported by a grant from the National Science Foundation.     

The involvement of thioredoxin and thioredoxin reductase in the dimethyl sulphoxide reductase system of Saccharomyces cerevisiae

Abstract Dimethyl sulphoxide (DMSO) reductase activity in crude extracts of Saccharomyces cerevisiae NCYC240 was stimulated by addition of thioredoxin, but not by addition of thioredoxin reductase. The activity was partially purified. DEAE-cellulose could be used to separate thioredoxin and its reductase (which bound to the column) from the terminal DMSO-reductase protein (which failed to bind). The highly unstable purified terminal reductase so obtained required both thioredoxin and thioredoxin reductase to reconstitute activity with either dithiothreitol (DTT) or NADPH as electron donor. Partially purified terminal reductase had an M _r of about 15000.     

Evidence for two different classes of redox-active cysteines in ribonucleotide reductase of Escherichia coli

The large subunit of ribonucleotide reductase from Escherichia coli contains redox-active cysteine residues. In separate experiments, five conserved and 2 nonconserved cysteine residues were substituted with alanines by oligonucleotide-directed mutagenesis. The activities of the mutant proteins were determined in the presence of three different reductants: thioredoxin, glutaredoxin, or dithiothreitol. The results indicate two different classes of redox-active cysteines in ribonucleotide reductase: 1) C-terminal Cys-754 and Cys-759 responsible for the interaction with thioredoxin and glutaredoxin; and 2) Cys-225 and Cys-439 located at the nucleotide-binding site. Our classification of redox-active cysteines differs from the location of the active site cysteines in E. coli ribonucleotide reductase suggested previously (Lin, A.-N. I., Ashley, G. W., and Stubbe, J. (1987) Biochemistry 26, 6905-6909).     

Role of the NADP/thioredoxin system in the reduction of alpha-amylase and trypsin inhibitor proteins

Thioredoxin, reduced either enzymatically with NADPH and NADP-thioredoxin reductase or chemically with dithiothreitol, reduced alpha-amylase and trypsin inhibitor proteins from several sources. Included were cystine-rich seed representatives from wheat (alpha-amylase inhibitors), soybean (Bowman-Birk trypsin inhibitor), and corn (kernel trypsin inhibitor). This system also reduced other trypsin inhibitors: the soybean Kunitz inhibitor, bovine lung aprotinin, and egg white ovoinhibitor and ovomucoid proteins. The ability of these proteins to undergo reduction by thioredoxin was determined by 1) a coupled enzyme activation assay with chloroplast NADP-malate dehydrogenase or fructose-1,6-bisphosphatase, 2) a dye reduction assay with 5',5'-dithiobis(2-nitrobenzoic acid), and 3) a direct reduction method based on the fluorescent probe, monobromobimane, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Reduction experiments with the seed proteins were carried out with thioredoxin from wheat germ (h-type) or Escherichia coli; the corresponding experiments with the animal trypsin inhibitors were carried out with thioredoxin from calf thymus or E. coli. In all cases, thioredoxin appeared to act catalytically; the reduced form of glutathione was without effect. When considered in conjunction with earlier results on purothionin (confirmed and extended in the current study), the new findings suggest that the NADP/thioredoxin system functions in the reduction of protein inhibitors of seeds and animal tissues. These results also raise the question of the occurrence of glutaredoxin in plants, as E. coli glutaredoxin was found to promote the reduction of some but not all of the proteins tested.     

Activation of class III ribonucleotide reductase by thioredoxin

Anaerobic ribonucleotide reductase provides facultative and obligate anaerobic microorganisms with the deoxyribonucleoside triphosphates used for DNA chain elongation and repair. In Escherichia coli, the dimeric alpha2 enzyme contains, in its active form, a glycyl radical essential for the reduction of the substrate. The introduction of the glycyl radical results from the reductive cleavage of S-adenosylmethionine catalyzed by the reduced (4Fe-4S) center of a small activating protein called beta. This activation reaction has long been known to have an absolute requirement for dithiothreitol. Here, we report that thioredoxin, along with NADPH and NADPH:thioredoxin oxidoreductase, efficiently replaces dithiothreitol and reduces an unsuspected critical disulfide bond probably located on the C terminus of the alpha protein. Activation of reduced alpha protein does not require dithiothreitol or thioredoxin anymore, and activation rates are much faster than previously reported. Thus, in E. coli, thioredoxin has very different roles for class I ribonucleotide reductase where it is required for the substrate turnover and class III ribonucleotide reductase where it acts only for the activation of the enzyme.     

Assimilatory sulfate reduction in Escherichia coli: identification of the alternate cofactor for adenosine 3'-phosphate 5'-phosphosulfate reductase as glutaredoxin.

The alternate cofactor (7004 cofactor) for Escherichia coli adenosine 3'-phosphate 5'-phosphosulfate (PAPS) reductase originally discovered in an E. coli mutant (tsnC 7004) lacking thioredoxin activity has now been purified and characterized. The tryptic peptide map of the 7004 cofactor is totally different from that of thioredoxin, indicating that the two proteins are unrelated in their primary structure. The 7004 cofactor has an amino acid composition different from that of thioredoxin but similar to that of glutaredoxin, a protein required for the glutathione-dependent deoxyribonucleotide formation by ribonucleotide reductase. Thus, the 7004 cofactor could not be a mutated form of thioredoxin, as was suspected earlier. Thioredoxin but not glutaredoxin is a substrate for thioredoxin reductase, but both thioredoxin and glutaredoxin can catalyze the dithiothreitol- or glutathione-dependent reduction of PAPS. On a molar basis, the dithiothreitol-coupled cofactor activity of thioredoxin is three- to fourfold higher that that of glutaredoxin. Comparison of the cofactor activities in the glutathione-coupled and the dithiothreitol-coupled PAPS reductase reaction shows that the cofactor activity of thioredoxin in the glutathione-coupled reaction is only 23% of that observed in the dithiothreitol-coupled reaction. However, in the case of glutaredoxin, cofactor activities are approximately the same in both the dithiothreitol- and glutathione-coupled reactions.