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 β-(7-amido-4-methylcoumarin) as substrate. Release of 7-amino-4-methylcoumarin was determined fluorometrically. At pH 7.5, K m = 93 μM, 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.
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