Enzymatic Synthesis of Glutamine in Higher Plants.

The synthesis of glutamine has been studied extensively with enzyme preparations from animal tissues and from bacteria (1, 2, 3, 9, 10). These studies have shown that in such organisms, glutamine may be formed directly from glutamic acid and ammonia, the energy for the synthesis being supplied as adenosine triphosphate (ATP). Little is known, however, of the occurrence or characteristics of similar reaction systems in higher plants. ELLIOTT (1) has reported the formation of glutamyl-hydroxamic acid in homogenates of Lupinus seedlings in the presence of added glutamate, hydroxylamine, ATP, and magnesium ions. This indicates that amide formation in these species probably proceeds in a manner analogous to its formation in other organisms. The present communication concerns studies on the general occurrence of glutamine synthesis in higher plants. Information is also presented on the nature of the synthetic system and its cellular localization. Homogenates of various seedlings, prepared in the manner described by MILLERD et al. (6) were dialyzed with shaking for three hours at 10 C against 0.01 M phosphate buffer (pH 7.0). For studies on cellular location of enzyme activity, undialyzed homogenates (from which heavy particles had been removed at 500 x g.) were centrifuged at 10,000 x g. as previously described (6) in order to obtain mitochondria. The supernatant from this operation was then centrifuged at 70,000 x g. to obtain the microsomal fraction. Acetone powders were prepared by grinding the plant material in five volumes of chilled acetone in a Waring Blendor at 10 C. The suspension was filtered with suction at 10 C, and the residue sucked dry in a stream of cold air. The operation was repeated, and the coarse powder was sifted through a screen. The resulting powder was stored in a desiccator at 10 C. Active extracts of acetone powders were prepared essentially in the manner described by SPECK (10). Unless otherwise indicated, the complete reaction system contained: 0.05 M potassium phosphate buffer (pH 7.0), 0.05 M sodium L-glutamate, 0.002 M sodium adenosine triphosphate, 0.02 M MgSO4, 0.04 M NH4Cl, 0.01 M cysteine, and one ml. of enzyme preparation in a total volume of