A radiometric assay for aspartoacylase activity in cultured oligodendrocytes
Section snippets
Materials
Solvents and unlabeled l-Asp were bought from Sigma (St.Louis, MO). Tris-HCl buffer, pH 8.0, from Digene (Beltsville, MD) and CaCl2 from Quality Biological (Gaithersberg, MD). l-[14C]Asp and l-[14C]Glu (specific activity of 207 mCi/mmol) were purchased from Amersham Pharmacia Biotech. Aluminum-supported flexible thin-layer chromatography (TLC) plates were purchased from Whatman Ltd, (Maidstone, Kent, England). Phosphor image exposure cassettes were bought from Molecular Dynamics. All other
Results and discussion
Fig. 1 shows the phosphor image of the TLC separation of the substrates [14C]NAA and [14C]NAG (Rf, 0.8) and their corresponding products l-[14C]Asp (Rf, 0.3) and l-[14C]Glu (Rf, 0.5). We have found that increasing the percentage of methanol in the solvent up to 30% increased the Rf in all cases, thus clearly separating both products from origin and also increasing the absolute separation between l-Asp and l-Glu from NAA and NAG, respectively. Comparison of the enzyme activity using the
Acknowledgements
This work was supported by a RO1 grant from NIH (NS39387) to M.A.A.N.
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An enzymatic fluorimetric assay for determination of N-acetylaspartate
2023, Analytical BiochemistryNon-genetic therapeutic approaches to Canavan disease
2016, Journal of the Neurological SciencesCitation Excerpt :However, it is not yet known how triheptanoin supplies substrates to oligodendrocytes, but triheptanoin was chosen based on its ability to produce TCA cycle intermediates while also providing substrates for lipogenesis. Since oligodendrocytes are an important source of CNS ASPA [71], if not the most important [34–36,72–74], repopulation of the brain of a CD patient with functional oligodendrocytes could potentially rectify much of the disease phenotype through the restoration of normal NAA metabolism. Neural stem cells have been successfully used to generate oligodendrocytes in the CD mouse model [75] and these oligodendrocytes expressed a myelin-specific enzyme indicative of myelin-producing ability, but clinical outcomes of such a treatment have yet to be investigated.
Evidence for mitochondrial and cytoplasmic N-acetylaspartate synthesis in SH-SY5Y neuroblastoma cells
2009, Neurochemistry InternationalN-acetylaspartate synthesis in the brain: Mitochondria vs. microsomes
2008, Brain ResearchCitation Excerpt :Protein standards also were chromatographed under similar conditions to ascertain the approximate molecular weight of the eluted enzyme. A radiometric assay with quantitation of the product by TLC-phosphor imaging technique as described earlier (Madhavarao et al., 2002, 2003) was followed with minor modifications. In brief, the assay was performed in same buffer {sorbitol phosphate buffer (pH 7.0) or PBS glycerol (pH 7.2)} containing [14C] l-Asp and unlabelled l-Asp at 1.06 mM with specific activity of ~ 6.7 mCi/mmol (13.5 × 106 CPM) and 1.0 mM acetyl CoA.
Mutational analysis of aspartoacylase: Implications for Canavan Disease
2007, Brain ResearchCitation Excerpt :Most mutations in this report yielded undetectable ASPA activity using a TLC-based assay and a fixed concentration of NAA near the published Km value (Namboodiri et al., 2000). Previous reports using different ASPA enzyme assays sometimes detected residual activity for various CD-associated ASPA mutations, likely as a result of longer enzyme–substrate incubations, higher substrate concentrations, and inherent background activity that is absent from the TLC-based assay (Madhavarao et al., 2002). With a partial homology model and a hypothetical catalytic mechanism for ASPA-mediated NAA hydrolysis, we can offer explanations for how several CD-associated mutations result in a loss of activity in vivo.