Glutamine Synthetase/Glutamine: α-Ketoglutarate Aminotransferase in Chloroplasts from the Marine Alga Caulerpa simpliciuscula

Abstract

The enzymic capacities for ammonia assimilation into amino acids have been investigated in chloroplasts from the siphonous green alga Caulerpa simpliciuscula (Turner) C. Ag. The results show that these chloroplasts differ from those of higher plants in having present simultaneously the enzymic capacities to permit assimilation of ammonia by two pathways.Glutamine synthetase (EC 6.3.1.2) activity at levels up to 4 jumoles per mg chlorophyll per hour were found in soluble extracts of the chloroplasts. Glutamine(amide):a-ketoglutarate aminotransferase (oxidoreductase ferredoxin) (EC 1.4.7.1) activity at levels up to 1.4 pmoles per mg chlorophyll per hour was detected by incubation of photosynthetically active chloroplasts either in light or with reduced ferredoxin. Together these enzymes provide the capacity for the conventional pathway of ammonium assimilation in chloroplasts via glutamine. A similar level of a glutamate dehydrogenase with an unusually low Km for ammonia which has been described previously in these chloroplasts provides the second potential pathway.Chloroplasts isolated from siphonous green algae have been shown recently to synthesize a different, and often wider, array of photosynthetic products from ''CO2 than higher plant chloroplasts (7,19). In this they resemble the chloroplasts isolated from Acetabularia where a similar range of products is synthesized by the isolated organelle (17). In this paper the studies of the metabolic competence of chloroplasts from siphonous green algae are extended to include the pathways of assimilation of ammonium into amino acids.In chloroplasts of higher plants ammonium is incorporated into amino acids via the two enzymic steps, glutamine synthetase (EC 6.3.1.2) followed by GOGAT2 (Jo). It has been suggested that the situation in chloroplasts isolated from the marine alga Caulerpa simpliciuscula is different, since they have been found to contain high levels of an NADP-dependent glutamate dehydrogenase, with an abnormally low apparent Km for ammonia of 0.4 to 0.7 mM (5). This enzyme could provide an alternative means of ammonium incorporation into amino acids in these algae.GOGAT activity has been demonstrated both in photosynthetic organisms which are more advanced in evolution than C. simpliciuscula and also in those which are more primitive (10, 11). We would expect that GOGAT activity should also be present in C. simpliciuscula unless this species represents a radical departure in evolution from other members of the plant kingdom. Here, we show that the chloroplasts of this siphonous green alga contain the enzymic capacities for both pathways of ammonium assimilation into amino acids. MATERIALS AND METHODSPlant Material. C. simpliciuscula plants were gathered at a depth of 1 to 2 m from the sea at Williamstown, Victoria and held in an illuminated tank of aerated and recirculating seawater on a 12-h light/12-h dark cycle at 16 C.Chloroplast Isolation. The chloroplast fraction was prepared from fronds of C. simpliciuscula by the method described prev...

Detection of glutamine synthetase activity in marine phytoplankton: optimization of the biosynthetic assay

“…This may apply in the case of the alga Caulerpa sirnpliciuscula (Gayler and Morgan, 1976;McKenzie et al. 1979).…”

“…Thus while GDH activity might be of prime in the case of Isochrysis galbana in which senescent importance in nitrogen assimilation in organisms such cells were assayed because they exhibited greater as Caulerpa simpliciuscula (Gayler and Morgan, 1976; levels of GS activity. McKenzie et al, 1979) and in Stichococcus bacillaris (Everest and Syrett, in press), it appears to play only a secondary role in marine phytoplankton. We report here the detection of glutamine synthetase activity in a wide variety of marine phytoplankton, all exhibiting much greater apparent affinity for scavenging ammonium ions than reported for GDH.…”

“…cells (Meister, 1974;Lea and Miflin, 1975;Falkowski and Rivkin, 1976;Meeks et al, 1977; HipBatch cultures were grown on artificial seawater kin and Syrett, 1977;Edge and Ricketts, 1978;Turpin medium (Pavlou, 1972). The only nitrogen source was and Harrison, 1978;McKenzie et al, 1979;Tischner NH,Cl at a concentration of 100 m. Table 1 lists the and Hiittermann, 1980; Sornarajah and Holdsworth, algal species cultured. The majority of these cultures 1981).…”

Glutamate Synthase Isoforms in Rice

“…It is now well accepted that glutamate synthase (glutamine:aketoglutarate aminotransferase; GOGAT2) catalyzes the second step of nitrogen assimilation into glutamate via glutamine in the presence of glutamine synthetase (EC 6.3.1.2) in bacteria (18,32), algae (10,14), nitrogen-fixing organisms (3,24), and higher plants (16).…”

Detection of glutamate synthase (GOGAT) activity in phytoplankton: evaluation of cofactors and assay optimization

“…NADH-GOGAT has been found in Chlamydomonas reinhardtu by Cullimore & Sims (1981a,b) and Marquez et al (1984) and in Euglena graciLis by Miyatake & Kitaoka (1981). Since Cuhmore & Sims (1981a) found that methyl viologen-GOGAT and NADH-GOGAT in C. reinhardtii appeared to be the same isozyme, the reported in vitro GOGAT activities mediated by the artificial electron donors methyl viologen in Caulerpa simpliciuscula (McKenzie et al 1979) and benzyl viologen in Platy-monas striata (Edge & hcketts 1978) may be indicative of NADH-GOGAT in these algal species. However, Marquez et al (1984) found that methyl viologen-GOGAT could correspond to both the NADH and ferredoxin forms of the enzyme.…”