Polarographic Study of Ammonia Assimilation by Isolated Chloroplasts

Abstract

Illuminated pea (Pisum sativum) chloroplasts catalyze (ammonia plus a-ketoglutarate [a-KGJ)-dependent 02 evolution at rates which are commensurate with other estimates of the flux of assimilated nitrogen (mean of eight determinations, 8.3 ,umole per mg chlorophyl per hour, SD 2.4). The reaction was usually initiated with 1 mm ammonia after preincubating chloroplasts in the presence of a-KG, ADP, pyrophosphate, and MgCl2.Progressive increases in ammonia concentration gave Vmax/2 at 0.2 mm (approximately) and Vmax at about 1 mm. Higher concentrations were inhibitory; at 7 mm the rate was again about Vmax/2. The highest ratio of 02 evolved per mol of ammonia supplied was 0.36.The (ammonia plus a-KG)-dependent reaction was inhibited by methionine sulfoximine, azaserine, and aspartate in the presence of amino-oxyacetate but not by amino-oxyacetate alone and not by Lglutamate. The rate of 02 evolution in the presence of 1 mm ammonia and 2.5 mM a-KG was increased only slightly by addition of 5 mM glutamine. Similarly, the rate of 02 evolution in the presence of 5 mM glutamine and 2.5 mM a-KG was increased only slightly by addition of 1 mm ammonia.The results are attributed to the incorporation of ammonia via glutamine synthetase and reductive transamination of the glutamine formed by photosynthetically coupled glutamate synthase using a-KG as the amino acceptor. Several lines of evidence rule out the possibility that photosynthetically coupled glutamate dehydrogenase is involved.It has long been known that glutamate is an early product of ammonia assimilation in plants (3,5). It now seems to be generally agreed that chloroplasts are the main site of reduction of nitrite to ammonia in leaf tissue (13,15). Glutamate dehydrogenase (NADPH-specific), glutamine synthetase, and glutamate synthase have been reported in chloroplasts suggesting that these organelles might also be an important site of ammonia assimilation.Illuminated chloroplasts catalyze the formation of glutamate from a-ketoglutarate (7) in the absence of exogenous ammonia. Ammonia (10 mM) had no significant effect on the reaction but the effect of lower concentrations was not reported. was attributed to photosynthetically coupled4 glutamate dehydrogenase (NADPH-specific). This enzyme is bound to the lamellae of Vicia faba chloroplasts (11). Glutamate dehydrogenase might account for ammonia assimilation in chloroplasts although the rate of glutamate formation (0.44 ,umol mg Chl-V hr-1) is low in comparison with the nitrite assimilation rates of chloroplasts of 6 to 12 ,umol mg Chl-' hr-1 (13) and 8 to 9,umol mg Ch-' hr-' (15). Further, the enzyme from Lactuca sativa chloroplasts has a low affinity for ammonia (10). Mitchell and Stocking (18) have recently shown that illuminated pea chloroplasts catalyze the formation of ['4C]glutamine from ['4C]glutamate in the presence of 1 mm ammonia at initial rates of 21 to 26 ,umol mg Chl-I hr-1. This suggests that glutamine synthetase, an enzyme known to be associated with chloroplasts (19, 23) and which has a hig...

Glutamine and Glutamate Transport in Cyanophora paradoxa

“…The glutamine and 2-0G-supported O 2 evolution described here for cyanelles was also described for chloroplasts and attributed to ferredoxin-dependent GOGAT (Anderson and Done, 1977;Woo and Osmond, 1982;Dry and Wiskich, 1983). 2-0G is transported into chloroplasts by a specific permease which shows some overlapping specificities with the dicarboxylate carrier but is clearly distinct from it.…”

Implication of the glutamine synthetase/glutamate synthase pathway in conditioning the amino acid metabolism in bundle sheath and mesophyll cells of maize leaves

“…In vitro Fd‐GOGAT assay showed that Fds reduced with NADPH via FNRs display a several‐fold higher ability to donate electrons to GOGAT (Table 2) than does photoreduced Fd [34]. In spite of the low FNR and Fd concentrations in BSCs (30 and 40 μ m ) [35,36], a close location of FNRs, Fds and Fd‐GOGAT on the thylakoid membrane [25,37] allows protein–protein complex formation essential for the GOGAT reaction [24].…”

Molecular aspects of plastid envelope biochemistry

“…During the course of photorespiratory NH3 reassimilation by intact chloroplasts, glutamate synthase and glutamine synthetase present in the stromal space established a selfsustaining system for ammonia incorporation where glutamine synthetase initiates the net synthesis of glutamate at the expense of 2-oxoglutarate [257]. Under these conditions, glutamate was continuously excreted from the chloroplasts in exchange for 2-oxoglutarate [258]. The marked effect of malate on the rate of glutamate excretion, observed by Woo and Osmond [259], led to the demonstration that 2-oxoglutarate is transported inward on a 2-oxoglutarate translocator and glutamate outward on a dicarboxylate translocator with malate as the counterion for both translocators in a cascade-like manner [260 -2621.…”

“…Under these conditions, glutamate was continuously excreted from the chloroplasts in exchange for 2-oxoglutarate [258]. The marked effect of malate on the rate of glutamate excretion, observed by Woo and Osmond [259], led to the demonstration that 2-oxoglutarate is transported inward on a 2-oxoglutarate translocator and glutamate outward on a dicarboxylate translocator with malate as the counterion for both translocators in a cascade-like manner [260 -2621.…”