Abstract: The aim was to study the extent to which leu-cine furnishes α-NH
2 groups for glutamate synthesis via branched-chain amino acid aminotransferase. The transfer of N from leucine to glutamate was determined by incubating astrocytes in a medium containing [
15N]leucine and 15 unlabeled amino acids; isotopic abundance was measured with gas chromatography-mass spectrometry. The ratio of labeling in both [
15N]glutamate/[
15N]leucine and [2-
15N]glutamine/[
15N]leucine suggested that at least one-fifth of all glutamate N had been derived from leucine nitrogen. At the same time, enrichment in [
15N]leucine declined, reflecting dilution of the
16N label by the unlabeled amino acids that were in the medium. Isotopic abundance in [
16N]-isoleucine increased very quickly, suggesting the rapidity of transamination between these amino acids. The appearance of
15N 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 α-ketoisocaproate (1 m
M), the ketoacid of leucine, the reaction readily ran in the “reverse” direction and intraastrocytic [glutamate] was reduced by ~50% in only 5 min. Extracellular concentrations of α-ketoisocaproate as low as 0.05 mM significantly lowered intracellular [glutamate]. The relative efficiency of branched-chain amino acid transamination was studied by incubating astrocytes with 15 unlabeled amino acids (0.1 m
M each) and [
15N]glutamate. After 45 min, the most highly labeled amino acid was [
15N]alanine, which was closely followed by [
15N]leucine and [
15N]isoleucine. Relatively little
15N was detected in any other amino acids, except for [
15N]serine. The transamination of leucine was ~17 times greater than the rate of [1-
14C]leucine oxidation. These data indicate that leucine is a major source of glutamate nitrogen. Conversely, reamination of a-ketoisocaproate, the ketoacid of leucine, affords a mechanism for the temporary “buffering” of intracellular glutamate.
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