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.
Arun
Peethambaran
P
Dept of Anatomy, Physiology and Genetics, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Rd, Bethesda, MD 20814, United States.
Moffett
John R
JR
Namboodiri
Aryan M A
AM
D000625:Aminooxyacetic Acid / Q000494:pharmacology; D001219:Aspartate Aminotransferases / Q000037:antagonists & inhibitors / Q000378:metabolism; D001224:Aspartic Acid / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000378:metabolism; D001921:Brain / Q000378:metabolism* / Q000648:ultrastructure; D002250:Carbon Radioisotopes / Q000378:metabolism; D002451:Cell Compartmentation / Q000502:physiology; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D003593:Cytoplasm / Q000378:metabolism* / Q000648:ultrastructure; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008293:Malates / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism* / Q000648:ultrastructure; D009474:Neurons / Q000378:metabolism* / Q000648:ultrastructure; D062907:Oxaloacetic Acid / Q000378:metabolism
D002250:Carbon Radioisotopes; D004791:Enzyme Inhibitors; D008293:Malates; D000625:Aminooxyacetic Acid; D062907:Oxaloacetic Acid; D001224:Aspartic Acid; C030298:malic acid; C000179:N-acetylaspartate; D001219:Aspartate Aminotransferases