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 redoxactive 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 μΜ DHA. Thus, the intracellular DHA concentration should be kept below 50 μΜ 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.
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. eoli protein. Minor thioltransferase activities (less than 10% of total leaf activity) have been observed in spinach chloroplasts which are probably doe to the presence of trypsin inhibitor and thioredoxins (TRf and TRm).
In our recent contribution [1] we questioned the role of dehydroascorbate (DHA) and DHA reductase as reliable indicators of oxidative stress in plants. Several lines of evidence were presented to substantiate that the measurement of DHA pools and DHA reductase activity in crude plant extracts is falsi¢ed by technical problems and by the fact that plant cells contain several redox-active proteins with unspeci¢c DHAreducing activity. We discussed whether the disproportionation of monodehydroascorbate (MDHA) leads to an appreciable formation of DHA in plant cells considering the known chemical and biochemical data of MDHA reductase and other MDHA-reducing components. Moreover, we questioned whether plant cells necessarily require a speci¢c DHA reductase and concluded that they probably do not possess a speci¢c DHA reductase at all. It is not surprising that these statements raise a dispute. Unfortunately, Foyer and Mullineaux [2] in their commentary try to counter our conclusions not by new facts but by remote arguments.First of all we did not suggest that DHA will not exist in plant cells at all. We outlined that it is improbable that signi¢cant amounts of DHA will accumulate, a conclusion drawn not only from ample biochemical evidence found in the literature [1] but from data showing that a steady-state concentration of 50 WM will substantially inhibit the regulatory action of plant thioredoxins essential for light-dependent regulation of processes such as CO P ¢xation, sulfate assimilation, and nitrate assimilation. Foyer and Mullineaux argue [2] that this observation was derived from assays not describing the situation in vivo because thioredoxin reductase was not added to regenerate reduced thioredoxin. They assume that the stromal enzymes regulated by the thioredoxin system require ongoing reduction to remain active. This would imply that in light the reductively activated target enzymes are permanently deactivated by oxidants and require repeated activation by thioredoxins in vivo. In the last 10 years knowledge about the action of thioredoxin has improved substantially enough (e.g. by demonstration of complex formation between thioredoxin and target enzymes) to indicate that the very opposite is the case. A detailed explanation is beyond the scope of this comment but the reader's attention is drawn to a recent review summarizing the biochemical action of thioredoxins [3].Among the known proteins possessing DHA reductase activity are thioredoxins, a Kunitz-type trypsin inhibitor, glutaredoxins (thiol transferases), and protein disul¢de isomerases. All these proteins are characterized by a very similar redoxactive site of the general amino acid sequence Cys-X-X-Cys. Reversible redox changes between the dithiol form and disul¢de form supply the reducing equivalents for DHA reduction. If we isolated one of these proteins using the common DHA reductase assay and did not know their original function in plant metabolism we, too, would regard it as DHA reductase. Many more enzymes containing Cys-X-X-Cys motifs migh...
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