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

Neurochem Int. 2009 Sep;55(4):219-25. doi: 10.1016/j.neuint.2009.03.003. Epub 2009 Mar 17.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

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

Substances

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