A radiometric assay for aspartoacylase activity in cultured oligodendrocytes.
Recent studies have shown that aspartoacylase (ASPA), the defective enzyme in Canavan disease, is detectable in the brain only in the oligodendrocytes. Studying the regulation of ASPA is central to the understanding the pathogenesis of Canavan disease and to the development of therapeutic strategies. Toward this goal, we have developed a sensitive method for the assay of ASPA in cultured oligodendrocytes. The method involves: (a) chemical synthesis of [14C]N-acetylaspartate (NAA) from L-[14C]Asp; (b) use of [14C]NAA as substrate in the assay; and (c) separation and quantitation of the product L-[14C]Asp using a TLC system. This method can detect as low as 10pmol of product and has been optimized for cultured oligodendrocytes. Thus, this method promises to be a valuable tool for understanding the biochemical mechanisms involved in the cell-specific expression and regulation of ASPA in oligodendrocytes.
Madhavarao
C N
CN
Department of Anatomy, Physiology and Genetics, USUHS, Bethesda, Maryland 20814, USA.
Hammer
J A
JA
Quarles
R H
RH
Namboodiri
M A A
MA
D000581:Amidohydrolases / Q000378:metabolism*; D000818:Animals; D001224:Aspartic Acid / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D001921:Brain / Q000201:enzymology; D002478:Cells, Cultured; D002855:Chromatography, Thin Layer; D005971:Glutamates / Q000138:chemical synthesis; D009836:Oligodendroglia / Q000201:enzymology*; D011874:Radiometry; D051381:Rats; D013053:Spectrophotometry
D005971:Glutamates; D001224:Aspartic Acid; C000179:N-acetylaspartate; D000581:Amidohydrolases; C019751:aspartoacylase; C016195:N-acetylglutamic acid