Characterization of the N-acetylaspartate biosynthetic enzyme from rat brain
Version of Record online: 21 JUL 2003
Journal of Neurochemistry
Volume 86, Issue 4, pages 824–835, August 2003
How to Cite
Madhavarao, C. N., Chinopoulos, C., Chandrasekaran, K. and Namboodiri, M. A. A. (2003), Characterization of the N-acetylaspartate biosynthetic enzyme from rat brain. Journal of Neurochemistry, 86: 824–835. doi: 10.1046/j.1471-4159.2003.01905.x
- Issue online: 21 JUL 2003
- Version of Record online: 21 JUL 2003
- Received March 31, 2003; accepted April 22, 2003.
- enzyme kinetics;
- high performance liquid chromatography;
- mitochondrial energetics;
- native gel electrophoresis;
- phosphor imaging;
- thin-layer chromatography
Aspartate N-acetyltransferase (Asp-NAT; EC 22.214.171.124) activity was found in highly purified intact mitochondria prepared by Percoll gradient centrifugation as well as in the three subfractions obtained after the sucrose density gradient centrifugation of Percoll purified mitochondria; citrate synthase was used as a marker enzyme for mitochondria. The proportion of recoverable activities of Asp-NAT and citrate synthase were comparable in mitochondrial and synaptosomal fractions but not in the fraction containing myelin. Asp-NAT was solubilized from the pellet of the rat brain homogenate (26 000 g for 1 h) for the recovery of maximum activity and partially purified using three protein separation methods: DEAE anion exchange chromatography, continuous elution native gel electrophoresis and size-exclusion high performance liquid chromatography. Asp-NAT activity and the optical density pattern of the eluted protein from size-exclusion column indicated a single large protein (∼670 kDa), which on sodium dodecyl sulfate–polyacrylamide gel electrophoresis showed at least 10 bands indicative of an enzyme complex. This seemingly multi-subunit complex Asp-NAT was stable towards ionic perturbations but vulnerable to hydrophobic perturbation; almost 95% of activity was lost after 10 mm 3-[(3-cholamidopropyl)dimethylammonia]-1-propanesulfonate (CHAPS) treatment followed by size-exclusion chromatography. Asp-NAT showed an order of magnitude difference in Km between l-aspartate (l-Asp, ∼0.5 mm) and acetyl CoA (∼0.05 mm). Asp-NAT showed high specificity towards l-Asp with 3% or less activity towards l-Glu, l-Asn, l-Gln and Asp-Glu. A model on the integral involvement of NAA synthesis in the energetics of neuronal mitochondria is proposed.