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 reduc-tase, nitrite reductase) were activated by light (I1). Such a light-mediated 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 METHODS In all experiments, synchronous cultures of Chlorella soroki-niana 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 GS-activity 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. RESULTS With 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 DTE enhance GS activity. The addition of large amounts of TR in the presence of 5 mm DTE results in an 8-fold activation (Table II). The maximum stimulated rate is similar to that obtained in vivo after 1 h of illumination (12). In further experiments, we were able to activate GOGAT in such extracts by similar treatments (Fig. 2). Here again, the incubation with TR alone did not stimulate the enzyme. However, in contrast to the results for GS, the application of DTE alone enhanced GOGAT activity. The combination of TR plus DTE was most effective with more than a 12-fold increase in the activity. If we subtract the DTE effect from this result, the pure TR effect can be calculated as a 4-fold stimulation of GOGAT activity. Our results indicate that the regulation of two key enzymes of nitrate metabolism (GS and GOGAT) is mediated by TR. In addition, we suggest that the effect of light on nitrate metabolism is based on an activation of GS/GOGAT via TR. The enzymes of the nitrate reducing system probably become substrate activated .