Dehydroascorbate reduction: the phantom remaining.
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Dehydroascorbic Acid↗
Biomedical subjects
I Häberlein
Publications and source records attributed to I Häberlein.
Sulfitolysis and thioredoxin-dependent reduction reveal the presence of a structural disulfide bridge in spinach chloroplast fructose-1,6-bisphosphatase.
A significant difference between cytosolic and chloroplastic fructose-1,6-bisphosphatase (FbPase) is an extra peptide in the middle of chloroplast FbPase which contains three additional cysteine residues. Sit-directed mutagenesis experiments have shown that at least two of these cysteine residues are involved in forming the regulatory disulfide bridge [Jacquot, J.-P. et al., FEBS Lett. 401 (1997) 143-147] which is the presupposition for the thioredoxin-dependent control of chloroplast FbPase activity. Here we report that each subunit of the FbPase contains an additional structural disulfide bridge which has been observed by combined application of thioredoxins and sulfitolysis. Observation of the structural disulfide bridges by sulfitolysis was only possible when the FbPase was already specifically reduced by the homologous thioredoxin species TRm. and TRf from spinach chloroplasts. Interestingly, the accessibility of the structural disulfide bridge for sulfite ions depends on the thioredoxin species engaged in the thioredoxin/FbPase complex.
Chloroplasts↗
Dehydroascorbate and dehydroascorbate reductase are phantom indicators of oxidative stress in plants.
In many physiological studies dehydroascorbate (DHA) reductase is regarded as one of the chloroplast enzymes involved in the protection against oxidative stress. Here, evidence is presented that plant cells do not possess a specific DHA reductase. The DHA reductase activities measured in plant extracts are due to side reactions of proteins containing redox-active dicysteine sites. Native gel electrophoresis combined with specific activity staining revealed three different proteins with DHA reductase activity in leaf and chloroplast extracts. These proteins have been identified as thioredoxins and trypsin inhibitors (Kunitz type) by Western blot analysis. The essential regulatory functions of thioredoxins in chloroplast metabolism are strongly inhibited in the presence of as little as 50 microM DHA. Thus, the intracellular DHA concentration should be kept below 50 microM but not all proteins with DHA reductase activity are effective enough for this purpose. A specific DHA reductase is frequently demanded as part of the enzymatic equipment to avoid oxidative stress. We argue that this is not necessary because in chloroplasts DHA does not accumulate to any significant extent due to the high activities of monodehydroascorbate reductase and of reduced ferredoxin.
Dehydroascorbic Acid↗
Kinetic evidence for protein complexes between thioredoxin and NADP-malate dehydrogenase and presence of a thioredoxin binding site at the N-terminus of the enzyme.
The kinetics of activation of NADP-malate dehydrogenase (MDH; EC 1.1.1.82) from soybean and spinach leaves by the chloroplast thioredoxins isolated from the same plants, by the corresponding storage forms of the soybean chloroplast thioredoxins from soybean seeds, and by the bacterial Escherichia coli thioredoxin have been studied. The Hill equation has been applied to evaluate the saturation kinetics. The observed variable thioredoxin saturation characteristics (Vmax 0.37-14.5 mumol NADPH min-1 mg enzyme-1; K0.5 0.15-1.33 microM; Hill coefficient h 0.90-3.04) indicate that the activation of NADP-MDH depends strongly on the individual thioredoxin used. Thus, thioredoxin action is not solely due to simple reductive activation of the NADP-MDH. Specific thioredoxin complex formation between thioredoxin and NADP-MDH must be included into the mechanism of the activation process. To study the regulatory consequences of the specific thioredoxin/NADP-MDH complexes we investigated the saturation kinetics of the substrates NADPH and oxaloacetate in presence of different concentrations of each individual thioredoxin species. The kinetic characteristics of the substrates (S0.5, Vmax, and Hill coefficients h) varied individually in response to the different thioredoxin species substantiating our model of thioredoxin/ NADP-MDH complex formation. Aminopeptidase-K-truncated pea NADP-MDH has been used to demonstrate that the N-terminal 37 amino residues are involved in providing a specific thioredoxin binding site. The fact that the versatile light-dependent regulation of numerous enzyme activities by only two thioredoxin species in chloroplasts cannot be accomplished without the formation of thioredoxin/target enzyme complexes is discussed in detail.
Bacterial Proteins↗
Completion of the thioredoxin reaction mechanism: kinetic evidence for protein complexes between thioredoxin and fructose 1,6-bisphosphatase.
The activation of chloroplast fructose 1,6-bisphosphatase from spinach and soybean leaves by the two chloroplast thioredoxins isolated from the same plants has been studied. The thioredoxin saturation characteristics (Vmax: 0.15-103.2 mumol Pi/min per mg enzyme; K0.5: 0.0048-0.84 microM; Hill coefficient n: 1.02-3.80) indicate that in addition to the reductive activation by thioredoxin specific complex formation between thioredoxin and fructose 1,6-bisphosphatase is responsible for fine regulation of the enzyme activity. This complex formation has been inserted into the thioredoxin mechanism and the physiological consequences discussed. Obviously, physiologically relevant investigations of the thioredoxin-dependent regulation of fructose 1,6-bisphosphatase activity can only be performed in homologous enzyme-thioredoxin combinations. Dithiothreitol and E. coli thioredoxin are no complete substitutes in regulatory studies.
Chloroplasts↗
Identification and localization of the first glutaredoxin in leaves of a higher plant.
Glutaredoxin(thioltransferase) has been identified and purified to homogeneity from spinach leaves. Its cytosolic localization was demonstrated by chromatographic and immunological analysis of extracts from isolated spinach chloroplasts and mitochondria, respectively. Spinach glutaredoxin shows a significant crossreactivity with antibodies raised against E. coli glutaredoxin and possesses a specific thioltransferase activity comparable to that of the E. coli protein. Minor thioltransferase activities (less than 10% of total leaf activity) have been observed in spinach chloroplasts which are probably due to the presence of trypsin inhibitor and thioredoxins (TRf and TRm).
Amino Acid Sequence↗
A novel-dehydroascorbate reductase from spinach chloroplasts homologous to plant trypsin inhibitor.
Dehydroascorbate reductase has been isolated from spinach chloroplasts and purified to apparent homogeneity. The N-terminal amino acid sequence of the enzyme is homologous to the Kunitz-type trypsin inhibitors from plant sources. It is shown that spinach DHA reductase and soybean trypsin inhibitor are both capable of reducing dehydroascorbate when in the reduced (thiol) form but acquire trypsin-inhibiting activity in the oxidized (disulfide) state. Reduced chloroplast thioredoxins also reduce dehydroascorbate.
Amino Acid Sequence↗
Structure requirements for disulfide bridge sulfitolysis of oxidized Escherichia coli thioredoxin studied by fluorescence spectroscopy.
Sulfitolysis of wild-type and four mutated Escherichia coli thioredoxins ([D26A]thioredoxin, [P34H]thioredoxin, [K36E]thioredoxin and endo-Arg33a-thioredoxin) has been investigated at millimolar concentrations of sulfite in the absence of protein-denaturing agents by fluorescence spectroscopy. Sulfitolysis of the single disulfide bridge of these proteins is associated with an increase in fluorescence emissions at 345 nm. Evaluation of the fluorescence emission spectra revealed that sulfitolysis of thioredoxins is a homogenous process. The reactivities of the thioredoxins are determined by negatively charged (Asp26) or positively charged (Lys36) amino acid residues near the active site disulfide bridge.
Amino Acid Sequence↗
Facile sulfitolysis of the disulfide bonds in oxidized thioredoxin and glutaredoxin.
Thioredoxins and glutaredoxins, in their oxidized form, possess a single disulfide bridge located on an edge of the small compact molecules. In contrast to most other disulfide-containing proteins, this S-S bridge is cleaved by millimolar concentrations of sulfite in the absence of protein denaturing agents at pH 7-8 and ambient temperature; however, the reaction is not quantitative. Sulfitolysis of Escherichia coli thioredoxin was found to be associated with an increase in fluorescence at 345 nm. A comparative study of sulfitolysis in 12 different thioredoxins and glutaredoxins of bacterial and plant origin has been made. Although they are all thought to be highly conserved in three-dimensional structure, their reactivities towards sulfite and the effects of 6 M guanidinium chloride (not affecting, or enhancing sulfitolysis) vary strongly in the series, with E. coli thioredoxin being less reactive and plant thioredoxins and E. coli glutaredoxin being more susceptible molecules. Contrary to expectation, reaction with sulfite is not generally correlated with the presence of negatively or positively charged amino acid residues near the disulfide loop but is determined by individual sequence and surface features in every single protein. These results confirm our hypothesis that thioredoxin sulfitolysis and inactivation [Würfel, M., Häberlein, I., Follmann, H. (1990) FEBS Lett. 268, 146-148] can occur in plant cells under physiological conditions and provide a biochemical rationale for the phytotoxicity of SO2.
Amino Acid Sequence↗
Non-redox protein interactions in the thioredoxin activation of chloroplast enzymes.
Thioredoxin derivatives lacking SH groups such as S,S'-dicarboxymethyl-, dicarboxamidomethyl-thioredoxin and cysteine----serine mutant protein are capable of activating chloroplast NADP malate dehydrogenase and fructose-bisphosphatase when added to enzyme assays together with suboptimal amounts of native thioredoxin. The modified thioredoxins alone are inactive. These findings indicate that protein-protein interactions play a significant role in addition to disulfide/thiol exchange reactions in the light-driven regulation of plant enzymes by the various plant thioredoxins.
Amino Acid Sequence↗
Inactivation of thioredoxin by sulfite ions.
Oxidized thioredoxin undergoes sulfitolysis of its single disulfide bond at low concentrations of sulfite ions and protein and in the absence of denaturing agents. The reaction, which has an optimum at pH 8, was studied using [35S]sulfite and E. coli thioredoxin as model. The product, thioredoxin-S-sulfonate, has a half-life of several hours in solution. It is unable to activate chloroplast NADP malate dehydrogenase. Thioredoxin sulfitolysis may therefore be a physiologically important factor in mediating the phytotoxic effects of sulfur dioxide in plants.
Bacterial Proteins↗
Unexpected specificity in the thioredoxin activation of fructose-bis-phosphatases from different plants.
Green seedlings of soy bean and wheat contain, like the plant seeds, multiple thioredoxin proteins which possess all typical thioredoxin properties but are inactive in the stimulation assay with spinach fructose-bis-phosphatase. However the pure proteins do have thioredoxin f activity when tested with homologous enzymes isolated from soy bean or wheat leaves, respectively, in the presence of Mg++. This new type of species specificity, unknown in all other in vitro assays of reduced thioredoxins, has to be considered in characterizing complete thioredoxin profiles in plants.
Bacterial Proteins↗
Thioredoxins: universal, yet specific thiol-disulfide redox cofactors.
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Animals↗