Metabolic Conversion of Amino Acids Loaded in the Vacuole of Chara australis Internodal Cells

Abstract

Vacuoles of internodal cells of Chara australis (or Chara corallina) were loaded with a 10 millimolar amount of various amino acids by a perfusion method and incubated under continuous light. After 20 to 24 hours, the cell sap was collected, and free amino acids in it and the rest of the cell (cytoplasm) were analyzed. The only amino acid metabolized completely was alanine. About 40 to 80% of the aspartic acid, glutamine, serine, and glycine were metabolized, whereas less than 30% of the threonine, asparagine, isoasparagine, isoleucine, phenylalanine, 'y-ami-nobutyric acid, lysine, and arginine were metabolized. The figure for glutamic acid fluctuated between 10 and 100%. The main metabolites of alanine were glutamine, glycine and ammonia, which accumulated in the vacuole. Alanine utilization was not affected by L-methionine-D,L-sulfox-imine or azaserine, but was strongly inhibited by aminooxyacetate. The cell extract contained enough alanine aminotransferase activity to account for the rate of alanine metabolism. Considerable amounts of intracellular free amino acids are located in the vacuoles of plants (1, 4, 9, 16, 21, 22, 25) and yeast (24). Recent studies with isolated Hevea vacuoles have indicated that lysine uptake is stimulated by added ATP (3, 8). With tonoplast membrane vesicles prepared from yeast vacuoles, Ohsumi and Anraku (14) and Kakinuma et al. (7) demonstrated that the accumulation of some basic amino acids in the vesicles depends on an active transport mechanism, an antiport with protons driven by a specific ATPase located in the tonoplast. Amino acids may also be derived from hydrolysis of proteins by proteases in the vacuole, which is assumed to be one of the important functions of this organelle (10, 1 1). The vacuolar free amino acids have been reported to be relatively inactive in a metabolic sense (5, 24). In addition, once free amino acids and sugars are confined in the vacuole, their efflux from this compartment is nearly zero when measured with vacuoles isolated from protoplasts (6, 20). However, these compartmentalized free amino acids are proW ably utilized in times of deficiency. The mobilization process of these vacuolar free amino acids which has not yet been studied experimentally in any detail, was the subject of the present research. We replaced the natural cell sap of internodal cells of Chara australis (or Chara corallina) with artificial salt solution containing various amino acids, and examined the fate of the