Evidence for mitochondrial and cytoplasmic N-acetylaspartate synthesis in SH-SY5Y neuroblastoma cells.

Abstract

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.

• Authors: Peethambaran Arun, John R Moffett, Aryan M A Namboodiri
• Journal: Neurochemistry international (Neurochem Int) Vol. 55 Issue 4 Pg. 219-25 (Sep 2009) ISSN: 1872-9754 [Electronic] England
• PMID: 19524112 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Carbon Radioisotopes
• Enzyme Inhibitors
• Malates
• Aminooxyacetic Acid
• Oxaloacetic Acid
• Aspartic Acid
• malic acid
• N-acetylaspartate
• Aspartate Aminotransferases

Topics

• Aminooxyacetic Acid (pharmacology)
• Aspartate Aminotransferases (antagonists & inhibitors, metabolism)
• Aspartic Acid (analogs & derivatives, biosynthesis, metabolism)
• Brain (metabolism, ultrastructure)
• Carbon Radioisotopes (metabolism)
• Cell Compartmentation (physiology)
• Cell Line, Tumor
• Chromatography, High Pressure Liquid
• Cytoplasm (metabolism, ultrastructure)
• Enzyme Inhibitors (pharmacology)
• Humans
• Malates (metabolism)
• Mitochondria (metabolism, ultrastructure)
• Neurons (metabolism, ultrastructure)
• Oxaloacetic Acid (metabolism)