Purification Of Aspnat

There are few reports on the cellular and sub-cellular localization of Asp-NAT per se other than those reported on its product molecule NAA, which is predominantly localized in neurons (see Moffett and Namboodiri, this volume).13,14 An earlier report on the mitochondrial localization of Asp-NAT was based on efflux studies of NAA on isolated brain mitochondria.11 Recently, it was convincingly shown to be localized to mitochondria by demonstrating that Asp-NAT activity in rat brain was present in the highly purified mitochondrial subfractions, including the outer mitochondrial membrane, the contact sites of outer and inner mitochondrial membranes and the inner mitochondrial membrane + matrix of the mitochondria12. However, a recent report indicated detection of Asp-NAT activity in a significant amount in the microsomal fraction isolated from rat brain homogenates.33

The radiometric assay developed by modification of the assay for aspartoacylase34 aided in the purification of Asp-NAT.12 The purification procedure of Asp-NAT (summarized in Fig. 2) involved solubilization of Asp-NAT activity by treating crude mitochondrial pellets in a buffer containing 10 mM CHAPS, and subsequently purifying the enzyme activity in four steps: 1) anion exchange chromatography, 2) native gel electrophoresis/continuous elution (Model 491 Prep Cell, BioRad), 3) HPLC size exclusion chromatography and 4) HPLC size exclusion re-chromatography.

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