Incorporation of 14C‐bicarbonate into the free amino acid pool during hyperosmotic stress in an intertidal copepod

Abstract

AbstractIn response to hyperosmotic stress, the harpacticoid copepod Tigriopus californicus rapidly accumulates high intracellular concentrations of free alanine and proline. Using 14C‐bicarbonate as a tracer, in vivo experiments were carried out to determine if pyruvate or phosphoenolpyruvate carboxylation function in the synthesis of carbon skeletons for free amino acid (FAA) synthesis. Label was incorporated into four FAA (aspartate, glutamate, proline, and alanine) during hyperosmotic stress. Relative specific activities of the FAA synthesized in vivo and in vitro enzyme activities suggest that malic enzyme (EC 1.1.1.40) carries out the carboxylation reaction. Incorporation of label into proline was extensive for at least 24 hr after transfer of adults acclimated to 50% seawater to 100% seawater; the proline pool showed relatively slow turnover following its accumulation (approximately 40% drop in specific activity in 48 hr) compared to the other FAA (complete loss of label in 24 hr). While bicarbonate fixation occurs in animals acclimated to both 50% and 100% seawater, label is observed in glutamate and aspartate but not proline. The results of these experiments suggest that the metabolic pathway by which bicarbonate label is incorporated into proline is activated by hyperosmotic stress and is effectively shut down following 24–48 hr of proline accumulation.