The thioredoxin h system: potential applications.

The thioredoxin h system has the specific capability to reduce intramolecular disulfide bonds of proteins, thereby modifying their tertiary structure. It is involved in many processes: in the activation or deactivation of enzymes and enzyme inhibitors and in the germination process. This system can be used to improve the breadmaking quality of wheat by strengthening the dough. It can also decrease the epitope accessibility, then modifying the response of the IgE immune system. Transgenic barley and wheat have been created to confirm the functionality of the NADP-dependent thioredoxin h system.

Glutathione reductase in wheat grain. 1. Isolation and characterization.

Durum wheat (Triticum durum, Desf.) endosperm of mature kernels contained a single form of glutathione reductase (GR); it appeared about the 18th day after anthesis while another isoform, present at the early stages of grain development, disappeared between the 20th and 30th days after flowering. The form that was present at grain maturity was isolated and characterized. It was composed of two monomers, each one having an apparent molecular mass of about 60 kDa. The K(m) values for NADPH and for GSSG were 3.7 and 9.1 microM, respectively, and the V(m) values for NADPH and for GSSG were 594 and 575 microkat.mg(-)(1) protein, respectively. The pH(i) of the enzyme was situated between pH 4.4 and 4.5. At a constant temperature of 25 degrees C, the optimum GR activity was found to be between pH 7.5 and 8.0. It was relatively resistant to high temperatures and was very resistant to very low temperatures.

Thioredoxin-linked mitigation of allergic responses to wheat.

Thioredoxin, a ubiquitous 12-kDa regulatory disulfide protein, was found to reduce disulfide bonds of allergens (convert S-S to 2 SH) and thereby mitigate the allergenicity of commercial wheat preparations. Allergenic strength was determined by skin tests with a canine model for food allergy. Statistically significant mitigation was observed with 15 of 16 wheat-sensitive animals. The allergenicity of the protein fractions extracted from wheat flour with the indicated solvent was also assessed: the gliadins (ethanol) were the strongest allergens, followed by glutenins (acetic acid), albumins (water), and globulins (salt water). Of the gliadins, the alpha and beta fractions were most potent, followed by the gamma and omega types. Thioredoxin mitigated the allergenicity associated with the major protein fractions-i.e, the gliadins (including the alpha, beta, and gamma types) and the glutenins-but gave less consistent results with the minor fractions, the albumins and globulins. In all cases, mitigation was specific to thioredoxin that had been reduced either enzymically by NADPH and NADP-thioredoxin reductase or chemically by dithiothreitol; reduced glutathione was without significant effect. As in previous studies, thioredoxin was particularly effective in the reduction of intramolecular (intrachain) disulfide bonds. The present results demonstrate that the reduction of these disulfide bonds is accompanied by a statistically significant decrease in allergenicity of the active proteins. This decrease occurs alongside the changes identified previously-i.e., increased susceptibility to proteolysis and heat, and altered biochemical activity. The findings open the door to the testing of the thioredoxin system in the production of hypoallergenic, more-digestible foods.

Thiocalsin: a thioredoxin-linked, substrate-specific protease dependent on calcium.

We describe a protease, named "thiocalsin," that is activated by calcium but only after reductive activation by thioredoxin, a small protein with a redox-active disulfide group that functions widely in regulation. Thiocalsin appeared to be a 14-kDa serine protease that functions independently of calmodulin. The enzyme, purified from germinating wheat grain, specifically cleaved the major indigenous storage proteins, gliadins and glutenins, after they too had been reduced, preferentially by thioredoxin. The disulfide groups of the enzyme, as well as its protein substrates, were reduced by thioredoxin via NADPH and the associated enzyme, NADP-thioredoxin reductase. The results broaden the roles of thioredoxin and calcium and suggest a joint function in activating thiocalsin, thereby providing amino acids for germination and seedling development.

Specific reduction of wheat storage proteins by thioredoxin h.

Gliadins and glutenins, the major storage proteins of wheat endosperm (Triticum durum, Desf. cv Monroe), were reduced in vitro by the NADP/thioredoxin system (NADPH, NADP-thioredoxin reductase and thioredoxin; in plants, the h type) from either the same source or the bacterium Escherichia coli. A more limited reduction of certain members of these protein groups was achieved with the reduced form of glutathione or glutaredoxin, a protein known to replace thioredoxin in certain bacterial and mammalian enzyme systems but not known to occur in higher plants. Endosperm extracts contained the enzymes necessary to reduce NADP by the oxidative pentose phosphate pathway (hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase). The gliadins and glutenins were also reduced in vivo during germination--an event that accompanied their proteolytic breakdown. The results suggest that thioredoxin, reduced by NADPH generated via the oxidative pentose phosphate pathway, functions as a signal in germination to enhance metabolic processes such as the mobilization of storage proteins and, as found earlier, the activation of enzymes.

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.

[Purification and characterization of two peroxidases from hard wheat]. / Purification et caractérisation de deux peroxydases de blé dur

Two peroxidases A and B were purified from a borate buffer extract (pH = 10,4) of durum wheat semolina (Triticum durum), var. Bidi 17, by chromatography on DEAE-cellulose, salting out by 3M ammonium sulphate and two chromatographies on CM-cellulose; specific activities of peroxidase A or B were increased 114 or 66 fold. Molecular weight, amino acid composition, absorption spectrum, pH optimum, thermal stability and KM values differentiate the two enzymes. Ion Ca++ was shown as an activator of both peroxidase activities; the presence of an inhibitor in the crude extract was demonstrated.