A Polarographic Study of Glutamate Synthase Activity in Isolated Chloroplasts

Abstract

Illuminated pea (Pisum sativum) chloroplasts actively catalyzed (glutamine plus .alpha.-ketoglutarate)-dependent O2 evolution (average of 12 preparations 10.6 .mu.mol mg chlorophyll per h). The reaction was specific for glutamine and .alpha.-ketoglutarate; concentrations of 0.2 mM .alpha.-ketoglutarate and 0.6 mM glutamine, respectively, effected half-maximum rates of O2 evolution. The reaction was inhibited by 3-(3,4-dichlorophenyl)-1-1-dimethylurea and did not occur in the dark. After osmotic shock chloroplasts did not catalyze O2 evolution. The reaction was inhibited by azaserine and glutamate but not by 10 mM ammonia, 2.5 mM methionine sulfoximine, or 5 mM amino-oxyacetate; addition of amino-oxyacetate together with aspartate inhibited O2 evolution. Arsenate (3 mM) enhanced O2 evolution. The highest molar ratio for O2 evolved per mole of .alpha.-ketoglutarate supplied was 0.40; the corresponding values for glutamine in the absence and presence of 3 mM arsenate were 0.20 and 0.24, respectively. The (glutamine plus .alpha.-ketoglutarate)-dependent O2 evolution is attributed to photosynthetically coupled glutamate synthase [EC 1.4.1.13] activity and the activity is sufficient to account for the assimilation of inorganic N. The low molar ratio for glutamine is discussed. Chloroplasts also catalyzed (aspartate plus .alpha.-ketoglutarate)-dependent O2 evolution but this reaction was inhibited by 5 mM amino-oxyacetate and it was insensitive to azaserine and methionine sulfoximine. This reaction was attributed to transaminase and photosynthetically coupled malate dehydrogenase activities.