hymecromone and Gangliosidoses

GM2-gangliosidosis B1 variant is thought to be a rare disorder with a wide geographical and ethnic distribution. We report the biochemical findings obtained in different specimens from a group of 11 B1 variant patients originating from the north of Portugal. The biochemical data obtained seem to indicate that only one of these patients is a genetic compound presenting a clinical and biochemical pattern similar to the majority of B1 variant patients described in the literature, but somewhat different from the profile presented by the other patients reported here, who are homozygous for the 'DN-allele'.

Topics: Acetylglucosamine; beta-N-Acetylhexosaminidases; Child; Child, Preschool; Electrophoresis, Cellulose Acetate; Fibroblasts; G(M2) Ganglioside; Gangliosidoses; Genetic Variation; Heterozygote; Homozygote; Humans; Hymecromone; Leukocytes; Lymphocytes; Portugal

4-Methylumbelliferyl-N-acetylglucosamine-6-sulfate (4MUGLc6S) which is known to be a specific substrate for human hexosaminidase A was used to determine enzymatic features of canine GM2-gangliosidosis. The enzyme activity using 4MUGlc6S in affected dog brain and liver was less than 20 to 30% of control tissues, whereas total 4-methylumbelliferyl beta-glucosaminidase activity in canine GM2-gangliosidosis was normal or elevated. However, when beta-hexosaminidase was fractionated by DEAE-Sepharose column chromatography, beta-hexosaminidase A like fraction in affected dog tissues was reduced to 20 to 30% of control. These data suggest that canine GM2-gangliosidosis is analogous to human juvenile.

Topics: Animals; beta-N-Acetylhexosaminidases; Chromatography, DEAE-Cellulose; Disease Models, Animal; Dog Diseases; Dogs; G(M2) Ganglioside; Gangliosidoses; Hexosaminidase A; Hymecromone; Isoenzymes

Topics: beta-N-Acetylhexosaminidases; Catalysis; Cell Line; Fibroblasts; G(M2) Ganglioside; Gangliosidoses; Hexosaminidase A; Humans; Hymecromone; Skin

Measurement of hexosaminidase A (Hex A) is an important clinical chemical procedure in the classification of GM2 gangliosidosis genotypes. We have synthesized a new substrate which may be useful in both the biochemical diagnosis of GM2 gangliosidosis and the detection of heterozygotes for the Tay-Sachs disease (TSD) allele. 4-Methylumbelliferyl-beta-D-N-acetylglucosamine-6-sulfate (4MUGS) was synthesized by sulfation of 4MU-beta-D-N-acetylglucosamine (4MUG) with chlorosulfonic acid and purified through gel filtration and ion-exchange chromatography. The structure of 4MUGS was verified by elemental analysis and NMR. Hex A is approximately 100 times more active toward 4MUGS than Hex B. The advantage of this increased specificity is that Hex A can be determined in a one-step procedure which allows separation of normal control serum values from those of obligate heterozygotes. Alternatively, assay values obtained using both substrates can be transformed by application of an empirical equation that allows the calculation of both Hex A and Hex B without the requirement of thermal fractionation. Lower values for % Hex A in serum have been obtained for Tay-Sachs homozygotes using the 4MUGS assay procedure. The results of Hex A assays on fibroblast cell strains obtained from Tay-Sachs homozygotes, variant forms of GM2 gangliosidosis and normal controls are also discussed.

Topics: beta-N-Acetylhexosaminidases; Cell Line; Drug Stability; Fibroblasts; Gangliosidoses; Genetic Carrier Screening; Genotype; Hexosaminidase A; Hexosaminidase B; Hexosaminidases; Homozygote; Hot Temperature; Humans; Hymecromone; Kinetics; Liver; Substrate Specificity; Tay-Sachs Disease; Umbelliferones

p-Nitrophenyl-6-sulfo-2-acetamido-2-deoxy-beta-D-glucopyranoside, which is known to be a specific substrate for human hexosaminidase A, has recently been used successfully for diagnosis of variants B and B1 of GM2-gangliosidosis (Fuchs et al. 1983; Kytzia et al. 1983; Li et al. 1983). However, it is hydrolyzed by hexosaminidase S as well and is therefore not suitable for detection of patients with variant 0, who reach the normal range of activity toward this substrate. Assay of ganglioside GM2 cleaving activity in fibroblast extracts in the presence of the natural GM2 activator protein reveals residual hexosaminidase A activities of less than 2% of normal controls in two infantile and up to 7.5% in two juvenile patients with variant 0.

Topics: Acetylglucosamine; Cells, Cultured; Fibroblasts; G(M2) Ganglioside; Gangliosidoses; Genetic Variation; Glucosamine; Hexosaminidases; Humans; Hymecromone; Isoelectric Focusing; Skin; Substrate Specificity

Kinetic studies of 4-methylumbelliferyl neuraminidase activity were carried out in cultured skin fibroblasts from patients with various disorders of neuraminidase deficiency. Cell extracts from two patients with dysmorphic type sialidosis of infantile onset, with isolated deficiency of neuraminidase activity, and three patients with dysmorphic type sialidosis of juvenile onset, with combined deficiency of neuraminidase and beta-galactosidase activities, demonstrated 7-12 times higher apparent Km values than those of normal controls (1.0-1.5 mmol/l as compared with 0.12-0.15 mmol/l). The apparent Ki values for N-acetylneuraminic acid and colominic acid were also increased in the dysmorphic type (7-15 and 7-11 times the normal values, respectively). In contrast, in the normomorphic type, normal apparent Km and Ki values were found for 4-methylumbelliferyl neuraminidase activity in fibroblasts from one patient with isolated neuraminidase deficiency and two patients with combined deficiency of neuraminidase and beta-galactosidase. The altered kinetics in the dysmorphic cases indicates a primary defect in neuraminidase with a secondary deficiency of beta-galactosidase in patients with combined deficiency. It is not clear if the primary defect in the normomorphic cases involves a defect in neuraminidase other than a Km defect or if neuraminidase or both neuraminidase and beta-galactosidase deficiencies are secondary to another defect as yet undetermined.

Topics: Cell Line; Fibroblasts; G(M1) Ganglioside; Gangliosidoses; Humans; Hymecromone; Kinetics; Lactose Intolerance; Mucolipidoses; Neuraminidase