P e d i a t r i c s , D e t r o i t . Current assays of hexosaminidase A (Hex A) a r e e i t h e r i n d i r e c t (heat-inactivation method) o r complex (GM2 ganglioside substrate).The finding i n Sanfilippo syndrome type D (N-acetylglucosamine-6-sulfatase deficiency) t h a t Hex A can bypass the blockage i n keratan s u l f a t e degradation by cleavage en bloc of B-N-acetylglucosamine-6-sulfate, led t o t h e present approach. 4-methylumbelliferyl d e r i v a t i v e s of 0-N-acetylglucosamine-6-sulfate (MUBGlcNAc-6-S) and 8-N-acetylgalactosamine-6-sulfate (MUBGalNAc-6-S) were prepared from t h e commonly used unsulfated d e r i v a t i v e s . Sera (n=5), leukocytes (n=3) and f i b r o b l a s t s (n=2) from p a t i e n t s with Tay-Sachs d i s e a s e (TSD) demonstrated markedly d e f i c i e n t act i v i t i e s toward both s u l f a t e d s u b s t r a t e s , 2-4% of c o n t r o l a c t i v it i e s (n=12), and samples from o b l i g a t e heterozygotes f o r TSD (n=4) had intermediate a c t i v i t y values, 43-62% of c o n t r o l a c t i v it i e s . Fibroblasts (n=2) from p a t i e n t s with Morquio syndrome type A (N-acetylgalactosamine-6-sulfatase deficiency) had normal act i v i t i e s toward both s u l f a t e d s u b s t r a t e s but the c h a r a c t e r i s t i c urinary excretion of chondroitin s u l f a t e i n t h i s d i s e a s e , indic a t e s t h a t t h e r e i s no analogy with Sanfilippo syndrome type D and Hex A is incapable of cleaving en bloc BGalNAc-6-S from t h e n a t u r a l s u b s t r a t e . I n c o n t r a s t t o n a t u r a l s u b s t r a t e s , t h e synt h e t i c s u l f a t e d s u b s t r a t e s a r e both s p e c i f i c f o r Hex A and thus, can provide d i r e c t and simple methods f o r diagnosis as well a s f o r a n t e n a t a l and c a r r i e r d e t e c t i o n of TSD. UDP-N-acetylglucosamine (UDP-GlcNAc) i s t h e donor of N-acetylglucosaminyl-1-phosphate (GlcNAcP) i n t h e r e a c t i o n catalyzed by GlcNAcP t r a n s f e r a s e , t h e enzyme d e f i c i e n t i n p a t i e n t s with I -c e l l d i s e a s e (ICD) and pseudo-Hur e r po dystrophy (PHPD). The use of f commercially a v a i l a b l e UDP-[ H o r "C]GlcNAc r a t h e r than the synt h e t i c a l l y made [B-~~PJUDP-G~CNAC was inadequate because of high background. We have overcome t h i s i n t h e assay of GlcNAcP t r a s f er a s e with a-methylmannoside acceptor by removal of f r e e [ 3~ o r 1 4~]~l c~~c which appeared t o be t h e major breakdown product. I n addition, t h e a-methylmannose-6-pho~pho-l-[~~ or 14~]GlcNAc product of t h e t r a n s f e r r e a c t i o n was then i s o l a t e d and following des a l t i n g could be used a s a s u b s t r a t e f o r the assay of aGlcNAc phosphodiesterase. Using these r e l a t i v e l y simple methods, deficiency of GlcNAcP t r a n s f e r a s e a c t i v i t y could be demonstrated i n f i b r o b l a s t s from p a t i e n t s with t h e c l a s s i c a l forms of I C D (n=4; l e s s than 4% of c o n t r o l a c t i v i t ...
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