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A deficiency of palmitoyl-protein thioesterase (PPT) was recently shown to be the primary defect in infantile neuronal ceroid lipofuscinosis (INCL). The available enzyme assays are complicated and impractical for diagnostic use. We have recently developed a new, fluorometric assay for PPT based on the sensitive fluorochrome 4-methylumbelliferone, requiring an overnight incubation to measure PPT. Now we have synthesized an analogue of this substrate which allows PPT determinations in 1 h. This improved PPT assay is simple, sensitive, and robust and will facilitate the definition of the full clinical spectrum associated with a deficiency of PPT. PPT activity was readily detectable in fibroblasts, leukocytes, amniotic fluid cells, chorionic villi, plasma, and cerebrospinal fluid from controls. PPT activity was profoundly deficient in these tissues and fluids from INCL patients. Similarly, a deficiency of PPT activity was demonstrated in patients with the variant juvenile NCL with GROD. These results show the feasibility of rapid pre- and postnatal diagnosis of INCL and its variants., (Copyright 1999 Academic Press.)

4-Methylumbelliferyl-alpha-D-N-sulphoglucosaminide (MU-alpha-GlcNS) was synthesized and shown to be a substrate for the lysosomal heparin sulphamidase. Sanfilippo A patients' fibroblasts (n = 42) and lymphocytes (n = 1) showed 0-3% of mean normal heparin sulphamidase activity; in total leukocytes from patients (n = 8) sulphamidase activity was clearly deficient. In fibroblasts from obligate heterozygotes for Sanfilippo A, the sulphamidase activity was reduced in 9 out of 10 cases. Heparin sulphamidase desulphates MU-alpha GlcNS to MU-alpha GlcNH2 and further hydrolysis during a second incubation is required to liberate 4-methylumbelliferone, which can be measured. Yeast alpha-glucosidase, which has low but sufficient alpha-glucosaminidase activity, was used to hydrolyse the reaction intermediate MU-alpha GlcNH2 to release 4-methylumbelliferone and free glucosamine.