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Plant Physiology 60:504-508 (1977)
© 1977 American Society of Plant Biologists

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Articles

Polarographic Study of Ammonia Assimilation by Isolated Chloroplasts 1

John W. Anderson2 and James Done3

a Department of Biochemistry, Rothamsted Experimental Station, Harpenden, Herts. AL52JQ, England

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 nitrogen (mean of eight determinations, 8.3 µmole per mg chlorophyll per hour, 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.

2 Permanent address: Botany Department, La Trobe University, Bundoora, Vic. 3083, Australia.

3 Permanent address: Biochemistry Department, University of Sydney, Sydney, N.S.W. 2006, Australia.

1 This work was conducted while the authors were on sabbatical leave from their respective universities.






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Copyright © 1977 by the American Society of Plant Biologists