Thioredoxin 2: Cleavage with Cyanogen Bromide

Holmgren, Arne; Reichard, Peter

doi:10.1111/j.1432-1033.1967.tb00125.x

Cited by 141 publications

(84 citation statements)

References 26 publications

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“…trx was purified from Escherichia coli cells by the procedure of Holmgren and Reichard [9]. Experimental details on the purification and the characterization of 3aa-trx and GT-trx were recently published [10].…”

Section: Methodsmentioning

confidence: 99%

Chloroplast fructose‐1,6‐bisphosphatase: Modification of non‐covalent interactions promote the activation by chimeric Escherichia coli thioredoxins

1996

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Although all thioredoxins contain a highly conserved amino acid sequence responsible for thiolldisulfide exchanges, only chloroplast thioredoxin-f is effective in the reductive stimulation of chloroplast fructose-l,6-bisphosphatase. We set out to determine whether Escherichia coli thioredoxin becomes functional when selected modulators alter the conformation of the target enzyme. Wild type and chimeric Escherichia coli thioredoxins match the chloroplast counterpart when the activation of chloroplast fructose-l,6-bisphosphatase is performed in the presence of fructose 1,6-bisphosphate, Ca 2+, and either trichloroacetate or 2-propanol. These modulators of enzyme activity do change the conformation of chloroplast fructose-l,6-bisphosphatase whereas bacterial thioredoxins remain unaltered. Given that fructose 1,6-bisphosphate, Ca 2+, and non-physiological perturbants modify non-covalent interactions of the protein but do not participate in redox reactions, these results strongly suggest that the conformation of the target enzyme regulates the rate of thioi/disulfide exchanges catalyzed by protein disulfide oxidoreductases.

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Section: Methodsmentioning

confidence: 99%

Chloroplast fructose‐1,6‐bisphosphatase: Modification of non‐covalent interactions promote the activation by chimeric Escherichia coli thioredoxins

1996

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“…Bovine serum albumin was from British Drug Houses. Thioredoxin [4] and thioredoxin reductase [S] were preparations from E. coli B. The glutathione analogues 2-4 were from previous studies [6].…”

Section: Introduction 2 Materials and Methodsmentioning

confidence: 99%

Glutathione derivatives as inhibitors of glutaredoxin and ribonucleotides reductase from Escherichia coli

et al. 1982

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“…coli B was prepared by the previously described "Method 2" [7] and had a specific activity value of 80-83 nmol thioredoxin per Peptide B, the N-terminal cyanogen bromide fragment of thioredoxin, was obtained as described previously [7]. Thioredoxin reductase [8] and ribonucleotide reductase [9,10] were preparations available in this laboratory.…”

Section: Methodsmentioning

confidence: 99%

“…coli B was prepared by the previously described "Method 2" [7] and had a specific activity value of 80-83 nmol thioredoxin per…”

Section: Methodsmentioning

confidence: 99%

Reversible Chemical Modification of the Tryptophan Residues of Thioredoxin from Escherichia coli B

Holmgren

1972

European Journal of Biochemistry

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Treatment of thioredoxin from Escherichia coli with formic acid saturated with HC1 by the method of Previero et al. (1967), resulted in a redshift of the ultraviolet spectrum of the protein consistent with chemical modification of both tryptophans to I-formyl-tryptophanyl residues, Concomitantly with the formylation the protein lost its redox activity in the enzymatic reactions with thioredoxin reductase and ribonucleotide reductase. Incubation of thioredoxin in formio acid alone had no effect on the spectrum or biological properties of the protein.Gel chromatographic experiments on Sephadex G-50 and G-75 showed that the formylated thioredoxin was eluted well apart from native thioredoxin and existed in the form of high molecular weight aggregates. This indicated that major conformational changes of thioredoxin accompanied the conversion of the hydrophobic tryptophan residues into the more hydrophilic I-formyl-tryptophans. Treatment of I-formyl-tryptophanyl thioredoxin a t pH 9.5 in 6 M guanidine hydrochloride for 16 h resulted in almost complete regeneration of the ultraviolet spectrum of native thioredoxin. Also regenerated was more than 50 of thioredoxin activity in the reaction with thioredoxin reductase. The results of this reversible chemical modification strongly indicat'e that the two tryptophan residues play a major role in maintaining the biologically active conformation of thioredoxin from E . coli.Thioredoxin from Escherichia coli consists of a single polypeptide chain of I08 amino acid residues [1,2]. The amino acid sequence of thioredoxin [ 2 ] shows that the functional part of this electron-transport protein consists of an intramolecular disulfide bridge arising from two half-cystine residues in positions 32 and 35. The disulfide of thioredoxin-S, (the oxidized form of thioredoxin) is reversibly reduced by NADPH in an enzymatic reaction with the specific enzyme thioredoxin reductase [3], according to reaction (1) :where thioredoxin-(SH), is the reduced form of thioredoxin.The two residues of tryptophan in thioredoxin, a t positions 28 and 31 are both close to the disulfide bridge in the primary structure [a]. Spectrofluorimetric measurements have shown that these tryptophan residues are characterized by unusually low Abbreviations. DTNB, 5,5'-dithiobis-(2-nitrobenzoic acid) ; thioredoxin-S, and thioredoxin-(SH,), the oxidized and reduced forms, respectively, of thioredoxin. fluorescence quantum yields when thioredoxin is in its oxidized form [4] ; reduction of thioredoxin-S, to thioredoxin-(SH),, however is accompanied by a three-fold increase in the quantum yield, indicating that one or both tryptophan residues participates in a conformational change of the protein [4].To study the role of tryptophan in structurefunction relationships in thioredoxin, specific and reversible chemical modification of tryptophan residues to I-formyl-tryptophanyl residues was performed by the technique of Previero et al.[5] and . I n the following paper the preparation of 1-formyl-tryptophanyl thioredoxin and its ...

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Thioredoxin 2: Cleavage with Cyanogen Bromide

Cited by 141 publications

References 26 publications

Chloroplast fructose‐1,6‐bisphosphatase: Modification of non‐covalent interactions promote the activation by chimeric Escherichia coli thioredoxins

Chloroplast fructose‐1,6‐bisphosphatase: Modification of non‐covalent interactions promote the activation by chimeric Escherichia coli thioredoxins

Glutathione derivatives as inhibitors of glutaredoxin and ribonucleotides reductase from Escherichia coli

Reversible Chemical Modification of the Tryptophan Residues of Thioredoxin from Escherichia coli B

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