Thioredoxin-mediated Activation Research Articles

Redox changes accompanying storage protein mobilization in moist chilled and warm incubated walnut kernels prior to germination

Alterations in the redox state of storage proteins and the associated proteolytic processes were investigated in moist-chilled and warm-incubated walnut (Juglans regia L.) kernels prior to germination. The kernel total protein labeling with a thiol-specific fluorochrome i.e. monobromobimane (mBBr) revealed more reduction of 29–32kDa putative glutelins, while in the soluble proteins, both putative glutelins and 41, 55 and 58kDa globulins contained reduced disulfide bonds during mobilization. Thus, the in vivo more reduced disulfide bonds of storage proteins corresponds to greater solubility. After the in vitro reduction of walnut kernel proteins pre-treated by N-ethyl maleimide (NEM) with dithioerythrethiol (DTT) and bacterial thioredoxin, the 58kDa putative globulin and a 6kDa putative albumin were identified as disulfide proteins. Thioredoxin stimulated the reduction of the H2O2-oxidized 6kDa polypeptide, but not the 58kDa polypeptide by DTT. The solubility of 6kDa putative albumin, 58 and 19–24kDa putative globulins and glutelins, respectively, were increased by DTT. The in vitro specific mobilization of the 58kDa polypeptide that occurred at pH 5.0 by the kernel endogenous protease was sensitive to the serine-protease inhibitor phenylmethylsulfonyl fluoride (PMSF) and stimulated by DTT. The specific degradation of the 58kDa polypeptide might be achieved through thioredoxin-mediated activation of a serine protease and/or reductive unfolding of its 58kDa polypeptide substrate. As redox changes in storage proteins occurred equally in both moist chilled and warm incubated walnut kernels, the regulatory functions of thioredoxins in promoting seed germination may be due to other germination related processes.

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.

The dimer contact area of sorghum NADP-malate dehydrogenase: role of aspartate 101 in dimer stability and catalytic activity

During thioredoxin-mediated activation of chloroplastic NADP-malate dehydrogenase, a homodimeric enzyme, the interaction between subunits is known to be loosened but maintained. A modeling of the 3D structure of the protein identified Asp-101 as being potentially involved in the association between subunits through an electrostatic interaction. Indeed, upon site-directed substitution of Asp-101 by an asparagine, the mutated enzyme behaved mainly as a monomer. The mutation strongly affected the catalytical efficiency of the enzyme. The now available 3D structure of the enzyme shows that Asp-101 is protruding at the dimer interface, interacting with Arg-268 of the neighbouring subunit.

The effect of high hydrostatic pressure on the modulation of regulatory enzymes from spinach chloroplasts.

High hydrostatic pressure enhanced the specific activity of regulatory enzymes of the Benson-Calvin cycle (fructose-1,6-bisphosphatase, glyceraldehyde-3-P dehydrogenase, phosphoribulokinase) which are modulated by the ferredoxin-thioredoxin system. High activity of chloroplast fructose-1,6-bisphosphatase required dithiothreitol, fructose 1,6-bisphosphate, and Ca2+. At 100 bar the A0.5 for fructose 1,6-bisphosphate (0.3 mM) was lower than that at 1 bar (1.5 mM), whereas similar variations of pressure did not alter the A0.5 for Ca2+ (55 microM). The response of chloroplast glyceraldehyde-3-P dehydrogenase exposed to 500 bar was a 4-fold increase in the NADP-linked activity; conversely, the NAD-dependent activity remained unchanged. The concerted action of high pressure and Pi (or ATP), both activators of chloroplast glyceraldehyde-3-P dehydrogenase, led to inactivation. On the other hand, the activity of phosphoribulokinase increased 10-fold when the enzyme was incubated at 1500 bar; the activation process was strictly dependent on the presence of dithiothreitol. At variance with these enzymes, bovine liver fructose-1,6-bisphosphatase, yeast glyceraldehyde-3-P dehydrogenase, and chloroplast ribulose 1,5-bisphosphate carboxylase, whose activities are not modulated by reduced thioredoxin, were inactivated by high pressure. The comparison of oligomeric enzymes revealed that the stimulation of specific activity by high pressure correlated with thioredoxin-mediated activation, and it did not depend on a particular subunit composition. Present results show that high pressure resembled thioredoxin, cosolvents, and chaotropic anions in its action on regulatory enzymes of the Benson-Calvin cycle. The comparison of physiological and non-physiological modulators suggested that thioredoxin-mediated modifications of noncovalent interactions is an important event in light-dependent regulation of chloroplast enzymes.

Molecular properties and thioredoxin-mediated activation of spinach chloroplastic NADP-malate dehydrogenase

There is no doubt that light-generated reducing equivalents can be used in C3 plants to perform a number of reactions occurring outside the chloroplasts [1-3]. Since the chloroplastic membrane is impermeable to NADPH and to reduced ferredoxin, it is currently believed that these reduction reactions in the cytoplasm involve the so-called C4dicarboxylic acid shuttle [4]. A specific malate translocator located on the chloroplastic membrane exports, outside the chloroplast, this dicarboxylic acid which is used in the cytosol to perform a number of reduction reactions [3]. NADPH formed during photosynthesis is used to reduce oxaloacetate to malate in the chloroplastic stroma. Chloroplastic malate dehydrogenase plays therefore an important role in the generation of reducing power used in reactions occurring in the cytoplasm. Moreover, crude extracts containing chloroplastic malate dehydrogenase are totally inactive in the dark and become active upon illumination. The effect of light is mimicked by a mixture of dithiothreitol and thioredoxin [5,6]. Surprisingly, chloroplastic NADP-malate dehydrogenase has never been purified to homogeneity. Therefore, nothing is known as to its structure, its reaction mechanism and the molecular bases of photoregulation by thioredoxin. Moreover one totally ignores whether several iso-forms of malate dehydrogenase exist in the chloroplast. The aim of this paper is to describe a purification procedure of chloroplastic NADP-malate dehydrogenase, to report some molecular properties of this enzyme as well as some features of its activation by dithiothreitol and thioredoxin.