Arginine Metabolism in Developing Soybean Cotyledons

Tracer kinetic experiments were performed using [ureido-(14)C] citrulline, [1-(14)C]ornithine, and isotope trapping techniques to determine if arginine is synthesized via the urea cycle in developing cotyledons of Glycine max (L.) Merrill. Excised cotyledons were injected with the (14)C-solution and incubated in sealed vials containing a CO(2) trap. The free and protein amino acids were analyzed using high performance liquid chromatography and arginine-specific enzyme-linked assays. In the (14)C-citrulline feeding experiment argininosuccinate was the most highly labeled compound after 5 minutes and it was the first compound to lose (14)C later in the time course. Carbon-14 was also recovered in free arginine, protein arginine, and CO(2) up to 4 hours after introduction of label. All of the (14)C in free and protein arginine could be accounted for in the C-6 position. Metabolism of (14)C-ornithine resulted in (14)C-incorporation into citrulline and free and protein arginine and the evolution of (14)CO(2). Citrulline was the most highly labeled compound after 15 minutes and was the first compound to reach a steady state level of (14)C. With the addition of 800 nanomoles unlabeled citrulline to the (14)C-ornithine feeding solution citrulline was the only compound labeled after 5 minutes and the steady state level of (14)C-citrulline increased 12-fold. The appearance of (14)C in free arginine and protein arginine was also delayed. In both (14)C-ornithine feedings all of the (14)C in free and protein arginine could be accounted for in the C-1 position. Together, the data support the reaction sequence: ornithine --> citrulline --> argininosuccinate --> arginine --> protein arginine.