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.

Both the alpha- and beta-anomers of 4-methylumbelliferyl-D-glucosaminide were synthesized and shown to be substrates for the lysosomal acetyl-CoA:glucosaminide N-acetyltransferase. Using the beta-anomer, fibroblasts and leukocytes from 11 different Sanfilippo C patients showed < 1% of mean normal N-acetyltransferase activity. Heterozygotes showed intermediate activities. The enzymatic liberation of the fluorochrome from 4-methylumbelliferyl-beta-D-glucosaminide requires the sequential action of the N-acetyltransferase and beta-hexosaminidase. Normal beta-hexosaminidase activity caused complete hydrolysis of the reaction intermediate 4-methylumbelliferyl-beta-D-N-acetylglucosaminide formed by the N-acetyltransferase. In cell extracts with a beta-hexosaminidase deficiency, however, a second incubation in the presence of excess beta-hexosaminidase is needed to avoid underestimation of the N-acetyltransferase activity.

Human-avian and human-mammalian influenza A virus reassortant clones with the neuraminidase (NA) gene of the A/USSR/90/77 (H1N1) strain and hemagglutinin (HA) genes of H3, H4 and H13 subtypes had been shown in an earlier publication to produce low HA yields in the embryonated chicken eggs. The low HA titers had been shown to be due, at least in part, to the formation of virion clusters at 4 degrees C; the clustering was removed by the treatment with bacterial neuraminidase [Rudneva et al., Arch. Virol (1993) 133: 437-450]. By serial passages of the reassortants in chick embryos non-aggregating variants were selected: the variants produced HA titers of the same order as A/USSR/90/77 parent virus. The assessment of the virus yields by the analysis of the partially purified virus preparations from fixed volumes of the allantoic fluid revealed that actual virion yields of the initial reassortants were lower than the yields of their passaged variants or of the parent viruses. The passaged variant of a reassortant possessing the HA gene of A/Duck/Ukraine/1/63 (H3N2) virus differed from the original (non-passaged) reassortant and from the parent A/Duck/Ukraine/1/63 virus in the reaction with a panel of monoclonal antibodies against H3 hemagglutinin. The data suggest that some HA-NA combinations may lead to an incomplete functional match between HA and NA and to the formation of low-yield reassortants, thus representing a possible limiting factor in the emergence of new HA-NA combinations in natural conditions.

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