[Show abstract][Hide abstract] ABSTRACT: Glutathione S-transferases (GSTs) (EC 126.96.36.199) are multifunctional proteins involved in such diverse intracellular events as primary and secondary metabolism, signaling and stress metabolism. In this study, we found a senescence-induced tau-class GST (SIGST) in senescent leaves of barley (Hordeum vulgare L.). The SIGST was purified 19-fold to homogeneity from initial crude extracts by three steps of chromatography with a yield of 5%. The purified SIGST had a GSH-conjugating activity and peroxidase (POD) activity at the same level of 1.7 micromol min(-1) mg protein(-1), although restricted substrate selectivity could be seen in POD activity. Barley SIGST is a slightly acidic protein with a molecular weight of 49 k and is composed of two subunits. The enzyme exhibited a single pH optimum at pH 8.3. The K(m) values were 0.285 mM for GSH and 0.293 mM for 1-chloro-2,4-dinitrobenzene. In most respects, the barley enzyme resembles those that have been reported from other higher plants. The SIGST gene was cloned from cDNA of senescent barley leaves. DNA sequence analysis shows that the cloned SIGST had only one base different from the barley embryo GST, ECGST. The obtained sequence indicates that SIGST is classified into the plant-specific tau class. mRNA expression analysis showed that in addition to senescence, SIGST was strongly induced by treatment with a herbicide and low temperature. The responses to these stresses suggest that SIGST may be involved at least partly in the secondary metabolism as an antioxidant and enhancement of enzymatic activity during senescence.
[Show abstract][Hide abstract] ABSTRACT: In the early steps of chlorophyll (Chl) degradation, the Mg-dechelation of chlorophyllide a (Chlide) to pheophorbide is known to be catalyzed by the so-called Mg-dechelatase. We previously demonstrated that the presence of a smaller metal-chelating substance (MCS) is required for the Mg-dechelation reaction using Chlide as the native substrate. This was further substantiated by this study in which Mg-dechelation activity in extracts from mature leaves of Chenopodium album was examined in complete fractions after gel filtration chromatography using an artificial substrate, Mg-chlorophyllin a (Chlin) and the native substrate, Chlide. A small-molecular-weight MCS and Mg-releasing proteins (MRPs) were present when Chlin was used as the substrate. However, only MCS had Mg-dechelation activity for the native substrate. Glutathione S-transferase (GST) was identified as one of the Mg-releasing proteins in addition to the previously known peroxidase (POD). Spontaneous release of Mg from Chlin was observed after the addition of a low concentration of hydrogen peroxide in the absence of MRPs, but not Chlide. These findings demonstrate that MCS plays a role in the catalysis of the Mg-dechelation reaction in the breakdown pathway of Chl. The release of Mg from Chlin by MRP may relate to the function of active oxygen species such as hydrogen peroxide. The relationship between Mg-release and the molecular plasticity of the artificial substrate compared to native substrate is discussed.
[Show abstract][Hide abstract] ABSTRACT: The Mg-dechelation activity in extracts from radish (Raphanus sativus L.) cotyledons was investigated using an artificial substrate, Mg-chlorophyllin a (Chlin) and the native substrate, chlorophyllide a (Chlide). In addition to a known a small molecular weight metal-chelating substance (MCS), Mg-releasing protein (MRP) was present when Chlin was used as the substrate. However, only MCS had Mg-dechelation activity with the native substrate. To examine the possibility of the dissociation of MRP into a protein moiety and a small molecular mass compound with an activity like MCS, extraction with low and high ionic strength buffers was carried out. No evidence was obtained that MCS is a moiety of MRP, however. Inhibitor studies showed that MCS and MRP had different susceptibilities to the inhibitors, especially to the chelators tiron and EDTA when Chlin was used as the substrate. Tiron had no effect on MRP, but it severely reduced MCS activity in both substrates. The activity of MRP increased during senescence, indicating the induction of MRP, while the activity of MCS was almost unchanged. These results suggest different reaction mechanisms by independent compounds. These findings suggest that MRP and MCS are present independently, and MCS is postulated to be a substance that catalyzes the Mg-dechelation reaction in the breakdown pathway of Chl, although MCS was not induced during senescence. The properties of MRP and MCS in relation to the small molecular mass substance obtained from strawberry fruit are also discussed.