INTERRELATIONSHIPS OF LEUCINE AND GLUTAMATE METABOLISM IN CULTURED ASTROCYTES

The aim was to study the extent to which leucine furnishes alpha-NH2 g roups for glutamate synthesis via branched-chain amino acid aminotrans ferase. The transfer of N from leucine to glutamate was determined by incubating astrocytes in a medium containing [N-15]leucine and 15 unla beled amino acids; isotopic abundance was measured with gas chromatogr aphy-mass spectrometry. The ratio of labeling in both [N-15]glutamate/ [N-15]leucine and [2-N-15]glutamine/[N-15]leucine suggested that at le ast one-fifth of all glutamate N had been derived from leucine nitroge n. At the same time, enrichment in [N-15]leucine declined, reflecting dilution of the N-15 label by the unlabeled amino acids that were in t he medium. Isotopic abundance in [N-15]- isoleucine increased very qui ckly, suggesting the rapidity of transamination between these amino ac ids. The appearance of N-15 in valine was more gradual. Measurement of branched-chain amino acid transaminase showed that the reaction from leucine to glutamate was approximately six times more active than from glutamate to leucine (8.72 vs. 1.46 nmol/min/mg of protein). However, when the medium was supplemented with alpha-ketoisocaproate (1 mM), t he ketoacid of leucine, the reaction readily ran in the ''reverse'' di rection and intraastrocytic [glutamate] was reduced by similar to 50% in only 5 min. Extracellular concentrations of alpha-ketoisocaproate a s low as 0.05 mM significantly lowered intracellular [glutamate]. The relative efficiency of branched-chain amino acid transamination was st udied by incubating astrocytes with 15 unlabeled amino acids (0.1 mM e ach) and [N-15]glutamate. After 45 min, the most highly labeled amino acid was [N-15]alanine, which was closely followed by [N-15]leucine an d [N-15]isoleucine. Relatively little N-15 was detected in any other a mino acids, except for [N-15]serine. The transamination of leucine was similar to 17 times greater than the rate of [1-C-14]leucine oxidatio n. These data indicate that leucine is a major source of glutamate nit rogen. Conversely, reamination of alpha-ketoisocaproate, the ketoacid of leucine, affords a mechanism for the temporary ''buffering'' of int racellular glutamate.