Polarographic Study of Ammonia Assimilation by Isolated Chloroplasts

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Abstract
Illuminated pea (Pisum sativum) chloroplasts catalyze (ammonia plus .alpha.-ketoglutarate [.alpha.-KG])-dependent O2 evolution at rates which are commensurate with other estimates of the flux of assimilated N (mean of 8 determinations, 8.3 .mu.mol/mg chlorophyll per h, SD 2.4). The reaction was usually initiated with 1 mM ammonia after preincubating chloroplasts in the presence of .alpha.-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 O2 evolved per mol of ammonia supplied was 0.36. The (ammonia plus .alpha.-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 L-glutamate. The rate of O2 evolution in the presence of 1 mM ammonia and 2.5 mM .alpha.-KG was increased only slightly by addition of 5 mM glutamine. Similarly, the rate of O2 evolution in the presence of 5 mM glutamine and 2.5 mM .alpha.-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 .alpha.-KG as the amino acceptor. Several lines of evidence rule out the possibility that photosynthetically coupled glutamate dehydrogenase is involved.