Yudkoff M, Daikhin Y, Lin Z P, Nissim I, Stern J, Pleasure D, Nissim I
Division of Metabolism, Children's Hospital of Philadelphia, University of Pennsylvania, School of Medicine 19104.
J Neurochem. 1994 Mar;62(3):1192-202. doi: 10.1046/j.1471-4159.1994.62031192.x.
The aim was to study the extent to which leucine furnishes alpha-NH2 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 15N label by the unlabeled amino acids that were in the medium. Isotopic abundance in [15N]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 alpha-ketoisocaproate (1 mM), the ketoacid of leucine, the reaction readily ran in the "reverse" direction and intraastrocytic [glutamate] was reduced by approximately 50% in only 5 min. Extracellular concentrations of alpha-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 mM 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 approximately 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 alpha-ketoisocaproate, the ketoacid of leucine, affords a mechanism for the temporary "buffering" of intracellular glutamate.
目的是研究亮氨酸通过支链氨基酸转氨酶为谷氨酸合成提供α-NH₂基团的程度。通过在含有[¹⁵N]亮氨酸和15种未标记氨基酸的培养基中培养星形胶质细胞来测定氮从亮氨酸向谷氨酸的转移；用气相色谱-质谱法测量同位素丰度。[¹⁵N]谷氨酸/[¹⁵N]亮氨酸和[2-¹⁵N]谷氨酰胺/[¹⁵N]亮氨酸中的标记比率表明，所有谷氨酸氮中至少五分之一来自亮氨酸氮。同时，[¹⁵N]亮氨酸中的富集度下降，这反映了培养基中未标记氨基酸对¹⁵N标记的稀释。[¹⁵N]异亮氨酸中的同位素丰度迅速增加，表明这些氨基酸之间转氨作用的快速性。缬氨酸中¹⁵N的出现较为缓慢。支链氨基酸转氨酶的测量表明，从亮氨酸到谷氨酸的反应活性比从谷氨酸到亮氨酸的反应活性高约6倍（8.72对1.46 nmol/分钟/毫克蛋白质）。然而，当培养基中添加亮氨酸的酮酸α-酮异己酸（1 mM）时，反应很容易朝“反向”进行，星形胶质细胞内的[谷氨酸]在仅5分钟内就减少了约50%。低至0.05 mM的细胞外α-酮异己酸浓度会显著降低细胞内[谷氨酸]。通过用15种未标记氨基酸（每种0.1 mM）和[¹⁵N]谷氨酸培养星形胶质细胞来研究支链氨基酸转氨作用的相对效率。45分钟后，标记程度最高的氨基酸是[¹⁵N]丙氨酸，紧随其后的是[¹⁵N]亮氨酸和[¹⁵N]异亮氨酸。除了[¹⁵N]丝氨酸外，在任何其他氨基酸中检测到的¹⁵N相对较少。亮氨酸的转氨作用比[¹-¹⁴C]亮氨酸氧化速率大约高17倍。这些数据表明亮氨酸是谷氨酸氮的主要来源。相反，亮氨酸的酮酸α-酮异己酸的再氨基化提供了一种对细胞内谷氨酸进行临时“缓冲”的机制。
