A Thioredoxin-Mediated Activation of Glutamine Synthetase and Glutamate Synthase in Synchronous Chlorella sorokiniana

Abstract

The effects of thioredoxin, dithioerythrol, and mixtures of both on enzymes involved in N metabolism of Chlorella sorokiniana have been studied. Glutamine synthetase, inactivated in vivo, was activated 8-fold by thioredoxin and dithioerythrol. By the same treatment, the activity of glutamate synthase was stimulated nearly 4-fold. Thus, two key enzymes of N metabolism were shown to be regulated via thioredoxin. The enzymes of the nitrate reducing system, i.e. nitrate reductase and nitrite reductase, were not affected by thiols. From these results, a model of NO3-metabolism is put forward which considers the regulating effect of light.The enzymes of the nitrate reducing system (i.e. nitrate reductase, nitrite reductase) were activated by light (I1). Such a lightmediated activation, without a de novo synthesis, was also demonstrated for glutamine synthetase and glutamate synthase (12). A thioredoxin-dependent activation of glutamine synthetase, which was partly purified from Chlorella and then inactivated in vitro, has been previously reported (8). In this paper, we concern ourselves with the role of thioredoxin in the light activation of enzymes related to nitrate assimilation. MATERIALS AND METHODSIn all experiments, synchronous cultures of Chlorella sorokiniana were used (high-temperature strain 211-8k of the algal collection of the Pflanzenphysiologisches Institut, University of Gottingen). Cultivation and synchronization procedures are described by Pirson et al (7). For our experiments, the cells were cultivated in a 7:17 (h/h) light:dark change in order to reach complete inactivation of the enzymes during the prolonged dark period.The preparation of the cell-free supernatant and the enzyme assays have been published previously (14). The values of GSactivity always relate to a synthetase assay.Activation Procedure. Thioredoxin, DTE,2 and TR plus DTE, respectively, were added to the enzyme assays in the given amounts. The reaction vessels were incubated at 30°C for 10 min. Then the enzyme reactions were started by the addition of the substrate. The thioredoxin used was identical to that previously prepared from spinach (TRf) (16). ' Supported by a grant from Deutsche Forschungsgemeinschaft to R. T.and A. S. 2 Abbreviations: DTE, dithioerythrol; TR, thioredoxin; NAR, nitrate reductase; NIR, nitrite reductase; GS, glutamine synthetase; GOGAT, glutamate synthase. RESULTSWith the onset of the illumination, the activity of NAR and NIR is stimulated 28-fold, and that of GS and GOGAT, 10-fold and 4-fold, respectively. These increases in activity are based on an activation of these enzymes already present in the cells and not on their de novo synthesis (11,12). A treatment of partly purified preparations from Chlorella autospores at the end of the synchronous lifecycle with TR, DTE, or TR plus DTE stimulated neither NAR nor NIR (Table I).In contrast, we succeeded in activating GS in those extracts, where the enzymes were present in an inactivated form in vivo. (Fig. 1). Only when presented together do TR and D...

Characterization of Glutamine Synthetase Isoforms from Chlorella

“…Since GS, and GS,, show very similar characteristics, it is concluded that the location of GS,, in the chloroplast rather than any alteration of its catalytic properties renders it more efficient in detoxifying NH3 than GS,, which is present in the cytosol (15). In chloroplasts, reduced thioredoxins are present which markedly activate GS in Chlorella (22). As previously discussed (2), it seems more logical that GS, might play a major role in the reassimilation ofphotorespiratory produced ammonia.…”

Glycolate Metabolism Is Under Nitrogen Control in Chlorella

“…Total GS activity was independent of applied 02 tensions, however. Recently it was observed that thioredoxin activates GS and glutamate synthase of C. sorokiniana (20). A difference in intracellular levels ofreduced thioredoxin might thus also result in a different capacity for the assimilation of NH3 produced during photorespiration.…”

“…Under these growth conditions the photorespiratory NH3 production is maximal. GS,, may be more effective in assimilating NH3 produced during photorespiration than GS, due to its higher affinity for glutamate (16) or due to its localization in the chloroplast where thioredoxins are present which activate the C. sorokiniana enzyme (20).…”

Thioredoxin-Linked Proteins Are Reduced during Germination ofMedicago truncatulaSeeds

“…Ketol acid reductoisomerase was previously reported as a potential target in cereal seeds (Wong et al, 2004b), phosphoglycerate dehydrogenase in spinach chloroplasts , and Gln synthetases in C. reinhardtii and Arabidopsis (Motohashi et al, 2001;Lemaire et al, 2004;Marchand et al, 2004;Yamazaki et al, 2004). The activity of both chloroplastic and cytosolic isoforms of Gln synthetase have long been known to be activated by Trx (Tischner and Schmidt, 1982;Florencio et al, 1993). The cytosolic isoform was found to undergo reduction in germinating M. truncatula seeds in this study.…”