title: A fluorometric assay for glycosylasparaginase activity and detection of aspartylglycosaminuria.
abstract: Recent experimental work on the mechanism of action of glycosylasparaginase suggests that the enzyme specifically reacts toward the L-asparagine or L-aspartic acid moiety of its substrates. Based on this, a new sensitive assay for glycosylasparaginase activity has been developed using L-aspartic acid beta-(7-amido-4-methylcoumarin) as substrate. Release of 7-amino-4-methylcoumarin was determined fluorometrically. At pH 7.5, Km = 93 microM, and as little as 1 ng of glycosylasparaginase could be detected with the assay. Hydrolysis of the substrate was inhibited by diazo-oxonorvaline, a specific inhibitor of glycosylasparaginase. In biological samples, the fluorometric assay is 40-100 times more sensitive than other published methods for glycosylasparaginase. This new assay enables a rapid enzymatic diagnosis of aspartylglycosaminuria--a genetic deficiency of glycosylasparaginase activity--with leukocyte and fibroblast samples.
authors.affiliation: Department of Clinical Chemistry, Kuopio University Hospital, Finland.
authors.forename: I T
authors.initials: IT
authors.lastname: Mononen
authors.forename: V M
authors.initials: VM
authors.lastname: Kaartinen
authors.forename: J C
authors.initials: JC
authors.lastname: Williams
mesh_terms: D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000652:urine; D054880:Aspartylglucosaminuria; D001227:Aspartylglucosylaminase / Q000032:analysis*; D005069:Evaluation Studies as Topic; D005347:Fibroblasts / Q000201:enzymology; D005470:Fluorometry / Q000379:methods* / Q000706:statistics & numerical data; D006801:Humans; D007962:Leukocytes / Q000201:enzymology; D008661:Metabolism, Inborn Errors / Q000175:diagnosis* / Q000201:enzymology / Q000652:urine; D012680:Sensitivity and Specificity
chemical_list: C000009:N-acetylglucosaminylasparagine; D001227:Aspartylglucosylaminase; D000117:Acetylglucosamine