The incorporation of tritiated water into amino acids in the presence of urea by white spruce seedlings in light and darkness

Abstract

White spruce seedlings containing urease were exposed to 0.16 M urea for 4 h in continuous light. Seedlings accumulated total soluble nitrogen as amides and arginine, and increased their content of bound amino acid nitrogen. In darkness, total soluble nitrogen declined and the increase of total bound amino acids was not as great as in light. In both treatments, the fate of tritiated water was examined by recovery of nonexchangeable tritium from amino acids. As urea was consumed, more tritium was recovered from seedlings in light than in darkness. In both treatments tritium followed the nitrogen of urea and was bound covalently, initially in glutamic acid, and subsequently wherever an α-keto acid was a precursor for the synthesis of the corresponding amino acid, viz. alanine and aspartic acid. In light, tritium was recovered mainly from glutamic acid, followed by glutamine, and to a lesser extent by γ-aminobutyric acid. In darkness, while glutamic acid was prominent initially, more radioactivity was recovered from γ-aminobutyric and glutamic acids compared to glutamine and to the light treatment. Glutamic acid was the main bound amino acid containing covalent tritium.The occurrence of tritium at the α-carbon of glutamic acid was supported by transfer of this tritium after decarboxylation to γ-aminobutyric acid, and by conversion of bound glutamate-3H to radioactive pyrrolidone carboxylic acid during acid hydrolysis of protein.Although urea nitrogen contributed to arginine synthesis in light, no tritium was found in arginine nor its precursors in the ornithine cycle until later, when nearly all amino acids were radioactive. This is consistent with the absence of covalent binding of tritium in ureido precursors leading to arginine biosynthesis, and supports the idea that tritium did not readily follow the carbon of urea into covalent linkage.