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Thioredoxins are 12-kDa proteins functional in the regulation of cellular processes throughout the animal, plant, and microbial kingdoms. Growing evidence with seeds suggests that an h-type of thioredoxin, reduced by NADPH via NADP-thioredoxin reductase, reduces disulfide bonds of target proteins and thereby acts as a wakeup call in germination. A better understanding of the role of thioredoxin in seeds as well as other systems could be achieved if more were known about the target proteins. To this end, we have devised a strategy for the comprehensive identification of proteins targeted by thioredoxin. Tissue extracts incubated with reduced thioredoxin are treated with a fluorescent probe (monobromobimane) to label sulfhydryl groups. The newly labeled proteins are isolated by conventional two-dimensional electrophoresis: (i) nonreducing͞reducing or (ii) isoelectric focusing͞reducing SDS/PAGE. The isolated proteins are identified by amino acid sequencing. Each electrophoresis system offers an advantage: the first method reveals the specificity of thioredoxin in the reduction of intramolecular vs. intermolecular disulfide bonds, whereas the second method improves the separation of the labeled proteins. By application of both methods to peanut seed extracts, we isolated at least 20 thioredoxin targets and identified 5-three allergens (Ara h2, Ara h3, and Ara h6) and two proteins not known to occur in peanut (desiccation-related and seed maturation protein). These findings open the door to the identification of proteins targeted by thioredoxin in a wide range of systems, thereby enhancing our understanding of its function and extending its technological and medical applications.peanut allergens ͉ desiccation-related protein ͉ seed maturation protein ͉ Ara h allergens ͉ proglycinin T here is a growing body of evidence that, as in photosynthesis, the regulatory protein thioredoxin (1-4) plays a role in heterotropic processes in plants. In this capacity, the disulfide group of a thioredoxin of the h-type is reduced by NADPH via the flavin enzyme, NADP-thioredoxin reductase (NTR) (1, 5, 6) (Eq. 1).Biochemical studies initiated a decade ago with wheat have provided evidence for a function of thioredoxin h in germination and seedling development. The results suggest that thioredoxin h, reduced via NTR with metabolically generated NADPH, acts early in the imbibed seed to initiate the mobilization of nitrogen and carbon in the endosperm, the major repository of storage protein and carbohydrate in cereals (7,8). The NADPH needed for this reduction can be generated enzymatically from carbohydrate stored in the endosperm via glucose 6-phophate and 6-phosphogluconate dehydrogenases (8).Through the reduction of intramolecular disulfide bonds (Eq. 2), thioredoxin h was shown to promote the degradation of major storage proteins, the inactivation of small proteins that inhibit amylolytic enzymes, and the activation of a novel calciumdependent substrate-specific protease (1,7,9,10 It has become clear that more complete information o...

The mobilization of storage proteins (glutelins) in germinating rice seeds was accompanied by an ordered sequential combination of proteolysis and reduction of disulfide groups. Mobilization was followed by application of non-reducing/reducing two dimensional-PAGE after monobromobimane labeling of the sulfhydryl groups of the proteins in intact seeds.

Many proteins undergo post-translational modification via well defined mechanisms such as acetylation, phosphorylation and glycosylation and thereby control a spectrum of biochemical processes. A growing body of evidence suggests that the reversible reduction of disulfide bonds also alters the structure and activity of proteins. Thioredoxin, a ubiquitous 12 kDa protein with a catalytically active disulfide active site (Cys-Gly-Pro-Cys), plays a central role in controlling the redox status of disulfide bonds in proteins that regulate a range of processes. Included are photosynthesis, seed germination, transcription, cell division, radical scavenging and detoxification. The ability to identify unknown functions of proteins of all types has been advanced by the emerging field of functional proteomics. In this brief review, we introduce the disulfide proteome as a tool that complements other methods for the comprehensive analysis of proteins. In so doing, the usefulness of applying this method for both in vitro and in vivo analyses is discussed for thioredoxin and other disulfide proteins, especially those occurring in plants.

The wide prevalence of celiac disease and wheat allergy has led to a growing demand for gluten-free foods. Rice proteins do not possess the viscoelastic properties typically found in gluten, thus making rice flour unsuitable for the production of yeast-leavened products. In the present study, we found that the addition of glutathione to rice batter improves its gas-retaining properties. Glutathione was found to prevent the formation of the disulfide-linked macromolecular protein barrier, which is reported to confer resistance to the deformation of rice batter in the baking process. Also, glutathione appeared to gelatinize rice starch at lower temperatures. Microstructure analyses of glutathione-added rice bread revealed it to have a perforated structure like wheat bread but with a smoother-looking surface. These data collectively suggest that glutathione facilitates the deformation of rice batter, thus increasing its elasticity in the early stages of bread baking and the volume of the resulting bread.

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Accumulating evidence suggests that redox regulations play important roles in a broad spectrum of biological processes. Recently, Yano et al. developed a disulfide proteome technique that comprehensively visualizes redox change in proteins. In this paper, using the disulfide proteome, we examined rice bran and identified fragments of embryo-specific protein and dienelactone hydrolase as putative targets of thioredoxin. Also, monitoring of the endogenous and recombinant effects of thioredoxin on rice bran proteins and supporting in vivo observations propose a mechanism of redox regulation in seed germination, in which thioredoxin activates cysteine protease with a concurrent unfolding of its substrate, the embryo-specific protein. Our findings suggest that thioredoxin controls the lifetime of specific proteins effectively by regulating the redox reactions coordinately. The model study demonstrates that the disulfide proteome technique is useful not only for identifying targets of thioredoxin, but also for clarify the detailed mechanism of redox regulation.

We have examined the viscosity effects of intramolecular excimer (IE) formation in 1,3-di-1-pyrenylpropane (DPP) at high pressures in various solvents. The formation is strongly and exclusively dependent on solvent viscosity, while it is insensitive to solvent polarity. The rates of the excimer formation estimated from fluorescence quantum yields are represented as a unique function of solvent viscosity. A hindered rotation model based on Kramers' theory was applied successfully. The more general results without specific interaction of solvent were obtained. The intrinsic activation energy (15-18 kJ/mol), the intrinsic activation volume (-2.5 cm3/mol), and the frequency for the top of the barrier of this IE formation were determined.Formation processes involving large-amplitude motion, such as intramolecular excimer (IE) formation in bichromophoric molecules in which bulky chromophores must twist with respect to the connecting bonds, should depend on the frictional forces exerted by solvents. For full understanding of these solvent effects, there are further questions to be answered. The first is whether the rates of formation are controlled exclusively by the viscosity of solvents; namely, whether the rates are equal at the same viscosity, independent of the means by which that viscosity value was produced, i.e., by changing the pressure, the temperature, or the solvent. The second is the question of what type of analytical formulation is possible for the viscosity dependence of the I E formation process and what type of model is applicable for describing the microscopic motions involved in the I E formation.We found in some bichromophoric compounds that the formation rates of their intramolecular excited states are really dependent on solvent viscosity.I4 As for the IE formation of 1,3-di-l-pyrenylpropane (DPP), Thistlethwaite et aL5 reported that it was not dependent on solvent viscosity, while Zachariasse et a1.6 argued against it. Thereafter, Offen et al.' reported a viscosity dependence that is inversely proportional to solvent viscosity, indicating a diffusion-controlled collision process. In our previous paper, we suggested from the study at high pressures8 that this formation is absolutely dependent on solvent viscosity and proposed that the hydrodynamic model of hindered rotation based on Kramers' theory may be applicable to the viscosity dependence.In most studies on the influence of solvent viscosity, a series of homologous solvent^^*^^ or mixed have been used to achieve viscosity variation. A possible problem in these approaches is that there may exist large local variations in the solvent shell interactions with the aromatic chromophores. Specific and/or steric interactions between solvent molecules and aromatic chromophores should affect the rotational transformation process and the stability of the excimer. The application of high hydrostatic pressure, on the other hand, makes it possible to minimize this problem. It achieves a large amount of continuous variation (1) Hara, K.; Arase, T.; Osugi, J...