Thioredoxin treatment increases digestibility and lowers allergenicity of milk.

BACKGROUND: By resisting digestion in the stomach, the major bovine milk allergen, beta-lactoglobulin, is believed to act as a transporter of vitamin A and retinol to the intestines. beta-Lactoglobulin has 2 intramolecular disulfide bonds that may be responsible for its allergic effects. OBJECTIVE: This study was carried out to assess the importance of disulfide bonds to the allergenicity and digestibility of beta-lactoglobulin. METHODS: beta-Lactoglobulin was subjected to reduction by the ubiquitous protein thioredoxin, which was itself reduced by the reduced form of nicotinamide adenine dinucleotide phosphate by means of nicotinamide adenine dinucleotide phosphate-thioredoxin reductase. Digestibility was measured with a simulated gastric fluid; results were analyzed by SDS-PAGE. Allergenicity was assessed with an inbred colony of high IgE-producing dogs sensitized to milk. RESULTS: As found for other proteins with intramolecular disulfide bonds, beta-lactoglobulin was reduced specifically by the thioredoxin system. After reduction of one or both of its disulfide bonds, beta-lactoglobulin became strikingly sensitive to pepsin and lost allergenicity as determined by skin test responses and gastrointestinal symptoms in the dog model. CONCLUSION: The results provide new evidence that thioredoxin can be applied to enhance digestibility and lower allergenicity of food proteins.

Morphological and biochemical responses of Bacillus subtilis to selenite stress.

When introduced into a chemically defined minimal medium supplemented with 1 mM sodium selenite (79 ppm Se(o)), Bacillus subtilis was found to undergo a series of morphological and biochemical adaptations. The morphological changes included the formation of "round bodies" associated with the detoxification of selenite to elemental selenium. Round bodies observed transiently were not apparent during balanced growth of cells adapted previously to selenite-containing medium. Under balanced growth conditions, cell structures similar to "round bodies", could be produced by treating cells with lysozyme. The selenite-induced structural alterations in cells were accompanied by an increase in the content of thioredoxin and the associated enzyme, NADP-thioredoxin reductase. The results suggest that the biovalence transformation of high levels of selenite may involve a dithiol system.

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.

Bioavailability of selenium accumulated by selenite-reducing bacteria.

The bioavailability of selenium (Se) was determined in bacterial strains that reduce selenite to red elemental Se (SeO). A laboratory strain of Bacillus subtilis and a bacterial rod isolated from soil in the vicinity of the Kesterson Reservoir, San Joaquin Valley, CA, (Microbacterium arborescens) were cultured in the presence of 1 mM sodium selenite (Na2SeO3). After harvest, the washed, lyophilized B. Subtilis and M. arborescens samples contained 2.62 and 4.23% total Se, respectively, which was shown to consist, within error, entirely of SeO. These preparations were fed to chicks as supplements to a low-Se, vitamin E-free diet. Three experiments showed that the Se in both bacteria had bioavailabilities of approx 2% that of selenite. A fourth experiment revealed that gray SeO had a bioavailability of 2% of selenite, but that the bioavailability of red SeO depended on the way it was prepared (by reduction of selenite). When glutathione was the reductant, bioavailability resembled that of gray SeO and bacterial Se; when ascorbate was the reductant, bioavailability was twice that level (3-4%). These findings suggest that aerobic bacteria such as B. subtilis and M. arborescens may be useful for the bioremediation of Se-contaminated sites, i.e., by converting selenite to a form of Se with very low bioavailability.

Physiological mechanisms regulating the conversion of selenite to elemental selenium by Bacillus subtilis.

We have demonstrated that the common soil bacterium, Bacillus subtilis, reduces selenite to an insoluble and much less toxic product--the red form of elemental selenium. Reduction was effected by an inducible system that appears to deposit elemental selenium between the cell wall and the plasma membrane. Glucose and sucrose supported selenite reduction. Although malate and citrate supported growth, no significant reduction of selenite occurred, indicating the importance of the redox state of the culture substrate. Selenite reduction in the millimolar concentration range (i.e., cultures supplemented with 1 mM selenite) was not affected by a ten-fold excess of nitrate or sulfate--compounds that serve as alternate electron acceptors and antagonize selenite reduction by anaerobic bacteria. Similarly, nitrite and sulfite did not significantly affect the rate or extent of selenite reduction. B.subtilis was able to grow and produce selenium (Se degree) at selenite concentrations ranging from 0.6 microM to 5 mM (50 ppb to 395 ppm selenium). At the lowest selenite concentration tested, 50 ppb selenium, B.subtilis removed 95% of the selenite from the liquid phase. The results suggest that selenite is reduced via an inducible detoxification system rather than dissimilatory electron transport. The findings establish the potential utility of B.subtilis for the bioremediation of selenite-polluted sites.

Amino acid sequences of heterotrophic and photosynthetic ferredoxins from the tomato plant (Lycopersicon esculentum Mill.).

Several forms (isoproteins) of ferredoxin in roots, leaves, and green and red pericarps in tomato plants (Lycopersicon esculentum Mill.) were earlier identified on the basis of N-terminal amino acid sequence and chromatographic behavior (Green et al. 1991). In the present study, a large scale preparation made possible determination of the full length amino acid sequence of the two ferredoxins from leaves. The ferredoxins characteristic of fruit and root were sequenced from the amino terminus to the 30th residue or beyond. The leaf ferredoxins were confirmed to be expressed in pericarp of both green and red fruit. The ferredoxins characteristic of fruit and root appeared to be restricted to those tissue. The results extend earlier findings in demonstrating that ferredoxin occurs in the major organs of the tomato plant where it appears to function irrespective of photosynthetic competence.

Thioredoxin-linked reductive inactivation of venom neurotoxins.

Thioredoxin, a 12-kDa protein with a catalytically active disulfide group, has recently been found to reduce intramolecular disulfide bonds in a variety of proteins. We now report that thioredoxin, reduced either enzymically with NADPH and NADP-thioredoxin reductase or chemically with dithiothreitol or lipoic acid, acts as a specific reductant of purified snake venom neurotoxins, a diverse group of disulfide proteins. Included were Bungarus multicinctus neurotoxins that act presynaptically (beta-bungarotoxin) or postsynaptically (alpha-bungarotoxin) as well as a postsynaptic neurotoxin from Laticauda semifasciata (erabutoxin b). We also observed a thioredoxin-specific reduction with other disulfide proteins of venom from Bungarus multicinctus, scorpion (Androctonus australis), and bee (Apis mellifera). Other cellular sulfhydryl agents, glutathione and glutaredoxin, were uniformly inactive. Thioredoxins from bacterial, plant, and animal sources were all active in neurotoxin reduction, but differed in effectiveness. Reduction of the neurotoxins by thioredoxin was accompanied by an increased susceptibility to tryptic proteolysis and a decrease of associated toxin activity: phospholipase A2 (beta-bungarotoxin, snake, and bee venoms) or acetylcholine receptor binding (alpha-bungarotoxin). These findings extend the function of thioredoxin to the reduction of a broad group of low-molecular-weight proteins, all containing intramolecular disulfide bonds. The loss of activity accompanying reduction raises the possibility that venoms may be detoxified by thioredoxin either as a defense mechanism or as a clinical antidote.

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.

Plant thioredoxin h: an animal-like thioredoxin occurring in multiple cell compartments.

Thioredoxin h has been purified to electrophoretic homogeneity from spinach roots using a procedure devised for leaves. The root thioredoxin (h2 form) differed from chloroplast and animal thioredoxins in showing an atypical active site (Cys-Ala-Pro-Cys) but otherwise resembled animal thioredoxin in structure. Sequence data for a total of 72 residues of spinach root thioredoxin h2 (about 69% of the primary structure) showed 43-44% identity with rabbit and rat thioredoxin. Analysis of cell fractions from the endosperm of germinating castor beans revealed that thioredoxin h occurs in the cytosol, endoplasmic reticulum, and mitochondria. The present findings demonstrate a similarity between plant thioredoxin h and animal thioredoxins in structure and intracellular location and raise the question of whether these proteins have similar functions.

Ferredoxin and ferredoxin-NADP reductase from photosynthetic and nonphotosynthetic tissues of tomato.

Ferredoxin and ferredoxin-NADP+ oxidoreductase (FNR) were purified from leaves, roots, and red and green pericarp of tomato (Lycopersicon esculentum, cv VFNT and cv Momotaro). Four different ferredoxins were identified on the basis of N-terminal amino acid sequence and charge. Ferredoxins I and II were the most prevalent forms in leaves and green pericarp, and ferredoxin III was the most prevalent in roots. Red pericarp of the VFNT cv yielded variable amounts of ferredoxins II and III plus a unique form, ferredoxin IV. Red pericarp of the Momotaro cv contained ferredoxins I, II, and IV. This represents the first demonstration of ferredoxin in a chromoplast-containing tissue. There were no major differences among the tomato ferredoxins in absorption spectrum or cytochrome c reduction activity. Two forms of FNR were present in tomato as judged by anion exchange chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. FNR II had a lower apparent relative molecular weight, a slightly altered absorption spectrum, and a lower specific activity for cytochrome c reduction than FNR I. FNR II could be a partially degraded form of FNR I. The FNRs from the different tissues of tomato plants all showed diaphorase activity, with FNR II being more active than FNR I. The presence of ferredoxin and FNR in heterotrophic tissues of tomato is consistent with the existence of a nonphotosynthetic ferredoxin/FNR redox pathway to support the function of ferredoxin-dependent enzymes.

Novel forms of ferredoxin and ferredoxin-NADP reductase from spinach roots.

Ferredoxin and the enzyme catalyzing its reduction by NADPH, ferredoxin-NADP reductase (ferredoxin-NADP+ oxidoreductase or FNR), were found to be present in roots of spinach (Spinacia oleracea). Localization experiments with endosperm of germinating castor beans (Ricinus communis), a classical nonphotosynthetic tissue for cell fractionation studies, confirmed that ferredoxin and FNR are localized in the plastid fraction. Both proteins were purified from spinach roots and found to resemble their leaf counterparts in activity, spectral properties, and complex formation, but to differ in amino acid composition and amino terminal sequence. The results indicate that the primary structures of the FNR and ferredoxin of spinach roots differ from that of the corresponding leaf proteins. Together with earlier findings, the present results provide evidence that nonphotosynthetic plastids, including those of roots, are capable of reducing ferredoxin with heterotrophically generated NADPH.

An NADP/thioredoxin system in leaves: purification and characterization of NADP-thioredoxin reductase and thioredoxin h from spinach.

An NADP/thioredoxin system, consisting of NADPH, NADP-thioredoxin reductase (NTR), and its thioredoxin, thioredoxin h, has been previously described for heterotrophic plant tissues, i.e., wheat seeds and cultured carrot cells. Until now there was no evidence for this system in green leaves. Here, we report the identification of protein components of the NADP/thioredoxin system in leaves of several species. Thioredoxin h and NTR, which were both recovered in the extrachloroplastic fraction, were purified to apparent homogeneity from spinach leaves. This represents the first time that NTR has been characterized from a plant source. Similar to that from bacterial and mammalian sources, spinach leaf NTR was a flavoprotein (Mr 68,000) composed of two subunits of identical molecular mass (Mr 33,000) that resembled Escherichia coli NTR immunologically. Spinach thioredoxin h existed in two forms (Mr of 13,500 and 12,000) and was highly specific for plant NTR. Thioredoxin h and NTR partially purified from spinach roots showed properties similar to their counterparts from leaves. Spinach cytosolic thioredoxin h differed from chloroplast thioredoxin m or f from the same source but was similar to thioredoxin h from wheat seed in immunological properties.

Thioredoxin from Rhodospirillum rubrum: primary structure and relation to thioredoxins from other photosynthetic bacteria.

Thioredoxin was isolated from a photosynthetic purple nonsulfur bacterium, Rhodospirillum rubrum, and its primary structure was determined by high-performance tandem mass spectrometry. The sequence identity of R. rubrum thioredoxin to Escherichia coli thioredoxin was intermediate to those of the Chlorobium thiosulfatophilum and Chromatium vinosum proteins. The results indicate that R. rubrum has an NADP-thioredoxin system similar to that of other photosynthetic purple bacteria.

A novel type of phosphofructokinase from plants.

A phosphofructokinase (PFK) has been purified to homogeneity from carrot roots as a large aggregated form (molecular weight greater than 5 million). The purified plant PFK, seemingly the cytosolic form, differed from its mammalian counterpart in a lower subunit molecular weight (60,000 verses 80,000), in being only sluggishly activated by fructose-2,6-bisphosphate, and in immunological properties. Similar to liver PFK, the purified carrot PFK could be dissociated by addition of 5 mM ATP to small and intermediate forms (respective molecular mass values of 2.4 X 10(5) and 6 X 10(5) Da). These small and intermediate forms could partially reassociate to the original large form in the presence of 5 mM Fru-6-P. Alkaline pH also effected the dissociation of the large and intermediate forms to the small form of PFK. All forms were present in significant amounts in freshly prepared carrot root extracts. The different forms of PFK showed characteristic pH activity profiles with pH optima of 8.6 (small form), 5.5 and 9.0 (intermediate form), and 7.0 and 8.5 (large forms). As alkaline pH (greater than or equal to approximately 8.5) dissociated the large and intermediate enzyme forms to yield the small form, it was concluded the "true" pH optima of the intermediate and large forms are pH 5.5 and 7.0, respectively. The pH optimum displayed by the intermediate and large forms in the alkaline region (pH 8.5-9.0) was considered to be due to their dissociation during assay. The different forms of PFK also had dissimilar regulatory properties, each showing a characteristic response to ATP, citrate, and Pi, but all were sensitive to inhibition by phosphoenolpyruvate and NADPH. Leaf cytosolic PFK, partially purified from spinach, showed similar properties. The results suggest that metabolite-dependent aggregation-disaggregation is a mechanism whereby plants regulate the activity of cytosolic PFK and the accompanying rate of glycolytic carbon flux.

Ferredoxin-thioredoxin reductase, an iron-sulfur enzyme linking light to enzyme regulation in oxygenic photosynthesis: purification and properties of the enzyme from C3, C4, and cyanobacterial species.

Ferredoxin-thioredoxin reductase (FTR), an enzyme involved in the light regulation of chloroplast enzymes, was purified to homogeneity from leaves of spinach (a C3 plant) and corn (a C4 plant) and from cells of a cyanobacterium (Nostoc muscorum). The enzyme is a yellowish brown iron-sulfur protein, containing four nonheme iron and labile sulfide groups, that catalyzes the activation of NADP-malate dehydrogenase and fructose 1,6-bisphosphatase in the presence of ferredoxin and of thioredoxin m and f, respectively. FTR is synonymous with the protein earlier called ferralterin. FTR showed an Mr of about 30,000 (determined by sedimentation equilibrium ultracentrifugation, amino acid composition, gel filtration, and gradient gel electrophoresis) and was composed of two dissimilar subunits (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). One of the FTR subunits from each source was similar both in Mr (about 13,000) and immunological properties, while the other subunit (of variable molecular weight) was characteristic of a particular organism. The similar subunit contained a disulfide group that was rapidly reduced by a dithiol (dithiothreitol) but not by monothiols (2-mercaptoethanol or reduced glutathione). Homogeneous FTR formed a tight noncovalent complex with ferredoxin on affinity columns. The basis for the structural variation in the different FTR enzymes remains to be determined.

Enzyme regulation in C4 photosynthesis: purification, properties, and activities of thioredoxins from C4 and C3 plants.

Procedures are described for the purification to homogeneity of chloroplast thioredoxins f and m from leaves of corn (Zea mays, a C4 plant) and spinach (Spinacea oleracea, a C3 plant). The C3 and C4f thioredoxins were similar immunologically and biochemically, but differed in certain of their physiochemical properties. The f thioredoxins from the two species were capable of activating both NADP-malate dehydrogenase (EC 1.1.1.37) and fructose-1,6-bisphosphatase (EC 3.1.3.11) when tested in standard thioredoxin assays. Relative to its spinach counterpart, corn thioredoxin f showed a greater molecular mass (15.0-16.0 kDa vs 10.5 kDa), lower isoelectric point (ca. 5.2 vs 6.0), and lower ability to form a stable noncovalent complex with its target fructose bisphosphatase enzyme. The C3 and C4 m thioredoxins were similar in their specificity (ability to activate NADP-malate dehydrogenase, and not fructose-1,6-bisphosphatase) and isoelectric points (ca. 4.8), but differed slightly in molecular mass (13.0 kDa for spinach vs 13.5 kDa for corn) and substantially in their immunological properties. Results obtained in conjunction with these studies demonstrated that the thioredoxin m-linked activation of NADP-malate dehydrogenase in selectively enhanced by the presence of halide ions (e.g., chloride) and by an organic solvent (e.g., 2-propanol). The results suggest that in vivo NADP-malate dehydrogenase interacts with thylakoid membranes and is regulated to a greater extent by thioredoxin m than thioredoxin f.

Contrasting modes of photosynthetic enzyme regulation in oxygenic and anoxygenic prokaryotes.

Enzymes that are regulated by the ferredoxin/thioredoxin system in chloroplasts--fructose-1,6-bis-phosphatase (FBPase), sedoheptulose-1,7-bisphosphatase (SBPase), and phosphoribulokinase (PRK)--were partially purified from two different types of photosynthetic prokaryotes (cyanobacteria, purple sulfur bacteria) and tested for a response to thioredoxins. Each of the enzymes from the cyanobacterium Nostoc muscorum, an oxygenic organism known to contain the ferredoxin/thioredoxin system, was activated by thioredoxins that had been reduced either chemically by dithiothreitol or photochemically by reduced ferredoxin and ferredoxin-thioredoxin reductase. Like their chloroplast counterparts, N. muscorum FBPase and SBPase were activated preferentially by reduced thioredoxin f. SBPase was also partially activated by thioredoxin m. PRK, which was present in two regulatory forms in N. muscorum, was activated similarly by thioredoxins f and m. Despite sharing the capacity for regulation by thioredoxins, the cyanobacterial FBPase and SBPase target enzymes differed antigenically from their chloroplast counterparts. The corresponding enzymes from Chromatium vinosum, an anoxygenic photosynthetic purple bacterium found recently to contain the NADP/thioredoxin system, differed from both those of cyanobacteria and chloroplasts in showing no response to reduced thioredoxin. Instead, C. vinosum FBPase, SBPase, and PRK activities were regulated by a metabolite effector, 5'-AMP. The evidence is in accord with the conclusion that thioredoxins function in regulating the reductive pentose phosphate cycle in oxygenic prokaryotes (cyanobacteria) that contain the ferredoxin/thioredoxin system, but not in anoxygenic prokaryotes (photosynthetic purple bacteria) that contain the NADP/thioredoxin system. In organisms of the latter type, enzyme effectors seem to play a dominant role in regulating photosynthetic carbon dioxide assimilation.

Enzyme regulation in C4 photosynthesis: mechanism of activation of NADP-malate dehydrogenase by reduced thioredoxin.

The mechanism of activation of thioredoxin-linked NADP-malate dehydrogenase was investigated by using 14C-iodoacetate and 14C-dansylated thioredoxin m, and Sepharose affinity columns (thioredoxin m, NADP-malate dehydrogenase) as probes to monitor enzyme sulfhydryl status and enzyme-thioredoxin interaction. The data indicate that NADP-malate dehydrogenase, purified to homogeneity from corn leaves, is activated by a net transfer of reducing equivalents from thioredoxin m, reduced by dithiothreitol, to enzyme disulfide groups, thereby yielding oxidized thioredoxin m and reduced enzyme. The appearance of new sulfhydryl groups that accompanies the activation of NADP-malate dehydrogenase appears to involve a structural change that is independent of the formation of a stable complex between the enzyme and reduced thioredoxin m. The data are consistent with the conclusion that oxygen promotes deactivation of NADP-malate dehydrogenase through oxidation of SH groups on reduced thioredoxin and on the reduced (activated) enzyme.