Abstract
Tracer kinetic experiments were performed using [ureido-14C] citrulline, [1-14C]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 14C-solution and incubated in sealed vials containing a CO2 trap. The free and protein amino acids were analyzed using high performance liquid chromatography and arginine-specific enzyme-linked assays. In the 14C-citrulline feeding experiment argininosuccinate was the most highly labeled compound after 5 minutes and it was the first compound to lose 14C later in the time course. Carbon-14 was also recovered in free arginine, protein arginine, and CO2 up to 4 hours after introduction of label. All of the 14C in free and protein arginine could be accounted for in the C-6 position. Metabolism of 14C-ornithine resulted in 14C-incorporation into citrulline and free and protein arginine and the evolution of 14CO2. Citrulline was the most highly labeled compound after 15 minutes and was the first compound to reach a steady state level of 14C. With the addition of 800 nanomoles unlabeled citrulline to the 14C-ornithine feeding solution citrulline was the only compound labeled after 5 minutes and the steady state level of 14C-citrulline increased 12-fold. The appearance of 14C in free arginine and protein arginine was also delayed. In both 14C-ornithine feedings all of the 14C 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.
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