N‐ACETYL‐ASPARTATE AMIDOHYDROLASE: PURIFICATION AND PROPERTIES¹

Abstract

Abstract— The enzyme in rat brain responsible for the de‐acetylation of N‐acetyl‐aspartic acid has been partially purified. In contrast to the enzyme from hog kidney which is stable at 70°C, it rapidly denatures above 57°C. The rat brain enzyme has the same Km for its substrate and the same solubility in ammonium sulphate solution as the hog kidney enzyme. Results of migration on starch gel electro‐phoreses and isoelectric focusing indicate a pI for the amidohydrolase of 5.1. A variety of potential substrates, modulators, and inhibitors have been examined.

N-Acetylaspartate

“…Biochemical studies showing ASPA occurrence predominantly in white matter of brain (McIntosh and Cooper, 1965; D' Adamo et al, 1973; provided early suggestions of a role in myelin formation and/or turnover, consonant with developmental data in the rat showing maximal enzyme activity and in situ hybridization at the peak of myelination. Initially described as a soluble enzyme (D' Adamo et al, 1973;1977), ASPA was also claimed to be membranebound as well ), a result supported by our findings . pointed to the requirement of detergent for solubilization as evidence of membrane association.…”

Cerebral metabolism in man after acute stroke: New observations using localized proton NMR spectroscopy

“…It is therefore plausible to assume an active degradative metabolic mechanism for NAA that occurs in injured neurons already in the first days (possibly hours) after onset ofinfarct. It may be hypothesized that amidohydrolase (9) which is thought to be compartmentalized or membrane-bound in healthy nervous tissue (10) may be released or activated in neuronal cell death in vivo. This is supported by a report that the NAA concentration decreased only very slowly in whole pieces of monkey brain, whereas it declined very rapidly (within 2 h) in incubated brain homogenates ( I I ) .…”

Extensive aspartoacylase expression in the rat central nervous system

“…ASPA has been reported to be present in the brain primarily as a soluble enzyme in the cytosol fraction (D′Adamo, Jr. et al, 1973; D′Adamo, Jr. et al, 1977; Klugmann et al, 2003; Madhavarao et al, 2004). However, other studies have provided evidence that some proportion of brain ASPA is associated with myelin (Chakraborty et al, 2001; Wang et al, 2007), and that detergent is required to dissociate this fraction of enzyme activity from membranes or myelin (Goldstein, 1976; Wang et al, 2007).…”

“…Aminoacylases 1 and 3 have relatively broad substrate specificity and act on a number of N-acetylated amino acids. ASPA, also known as aminoacylase 2, acts to deacetylate only one acetylated amino acid, namely N-acetylaspartate (NAA) (D′Adamo, Jr. et al, 1977; Madhavarao et al, 2003). ASPA hydrolyzes NAA into free acetate and aspartate (Kaul et al, 1993; Zeng et al, 2002) and is expressed strongly in a number of tissues including the brain and kidney (Birnbaum, 1955; Madhavarao et al, 2004; Hershfield et al, 2006).…”