N-acetylaspartate synthetase
cytoplasm
Evidence for mitochondrial and cytoplasmic N-acetylaspartate synthesis in SH-SY5Y neuroblastoma cells.
N-acetylaspartate (NAA) is synthesized predominantly in neurons, and brain homogenate subfractionation studies suggest that biosynthesis occurs at both microsomal (cytoplasmic) and mitochondrial sites. We investigated NAA synthesis in SH-SY5Y human neuroblastoma cells using distinct metabolic precursors that are preferentially metabolized in mitochondria and cytoplasm. Incorporation of (14)C-aspartate and (14)C-malate into NAA was examined in the presence and absence of an inhibitor (aminooxyacetic acid, AOAA) of aspartate aminotransferase (AAT), a central enzyme involved in the biosynthesis of aspartate in mitochondria, and degradation of aspartate in the cytoplasm. AOAA increased the incorporation of (14)C-aspartate into NAA, reflecting direct aspartate-->NAA synthesis in an extramitochondrial location. As expected, AOAA decreased incorporation of (14)C-malate into NAA, reflecting NAA synthesis in mitochondria via the malate-->oxaloacetate-->aspartate-->NAA pathway. When (14)C-malate was used as substrate, the (14)C-NAA/(14)C-aspartate ratio was over 20-fold higher than the ratio obtained with (14)C-aspartate. Because NAA can only be synthesized from aspartate, the higher (14)C-NAA/(14)C-aspartate (product/substrate) ratio obtained with (14)C-malate suggests greater NAA biosynthetic activity. We conclude that NAA biosynthesis occurs in both the cytoplasm and mitochondria of SH-SY5Y cells, and that the contribution from the mitochondrial compartment is quantitatively larger than that in the extramitochondrial compartment.