Ganglioside GM2 N-acetyl-beta-D-galactosaminidase and asialo GM2 (GA2) N-acetyl-beta-D-galactosaminidase; studies in human skin fibroblasts.
Ganglioside GM2 and its asialo-derivative, GA2 were radiolabeled in their N-acetyl-D-galactosaminyl moieties by oxidation with galactose oxidase and reduction with tritiated sodium borohydride. Specific activities of 6 X 10(4) dpm/nmol (GM2) and 1.8 X 10(6) dpm/nmol (GA2) were achieved. About 98% of the label was in N-acetyl-D-galactosamine. Using these substrates, an assay was developed for GM2-N-acetyl-beta-D-galactosaminidase (E.C.3.2.1.30) and GA2-N-acetyl-beta-D-galactosaminidase (E.C.3.2.1.30) activities in human cultured skin fibroblasts. The products of the GM2 cleaving reaction were identified as N-acetylgalactosamine and ganglioside GM3. Both GM2 and GA2 cleaving activities were stimulated about 5-fold by purified sodium taurocholate, and this stimulation was inhibited by neutral detergents, lipids and albumin at low concentrations. Addition of various salts, reducing agents and a protein activator factor from human liver of Li et al. (1973) did not stimulate GM2-N-acetyl-beta-D-galactosaminidase activity beyond that found with sodium taurocholate. Under optimal conditions, control fibroblast supernates cleaved ganglioside GM2 at a rate of 3.7 nmol/mg protein/h compared to 1100 for GA2-N-acetyl-beta-D-galactosaminidase and 4700 for 4-methylumbelliferyl-N-acetyl-beta-D-glucosaminidase. Supernates from two patients with Tay-Sachs disease had markedly reduced activity levels for GM2-N-acetyl-beta-D-galactosaminidase but not for the other two substrates. Supernates from two patients with Sandhoff's disease had reduced activities for all three substrates. A supernate from one patient with juvenile GM2 gangliosidosis cleaved GM2 at a somewhat faster rate than those from Tay-Sachs or Sandhoff's patients. Two healthy adult women with markedly reduced hexosaminidase A activities using 4MU-N-acetyl-beta-D-glucosaminide as substrate had approximately half-normal activities using GM2 as substrate. A patient with the Tay-Sachs phenotype but with a partial deficiency of hexosaminidase A using the 4-MU substrate had a profound deficiency using GM2 as substrate. In such unusual hexosaminidase mutants, assays using GM2 as substrate are better indicators of phenotype than those using synthetic substrates.
D000328:Adult; D000418:Albumins / Q000494:pharmacology; D001894:Borohydrides / Q000494:pharmacology; D002478:Cells, Cultured; D002855:Chromatography, Thin Layer; D003902:Detergents / Q000494:pharmacology; D005260:Female; D005347:Fibroblasts / Q000201:enzymology; D005678:G(M2) Ganglioside / Q000031:analogs & derivatives / Q000378:metabolism*; D005692:Galactose Oxidase / Q000494:pharmacology; D005732:Gangliosides / Q000378:metabolism*; D005733:Gangliosidoses / Q000201:enzymology; D006596:Hexosaminidases / Q000172:deficiency / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007553:Isotope Labeling; D008064:Lipidoses / Q000201:enzymology; D008055:Lipids / Q000494:pharmacology; D012867:Skin / Q000201:enzymology*; D013656:Taurocholic Acid / Q000494:pharmacology
D000418:Albumins; D001894:Borohydrides; D003902:Detergents; D005732:Gangliosides; D008055:Lipids; D005678:G(M2) Ganglioside; D013656:Taurocholic Acid; D005692:Galactose Oxidase; D006596:Hexosaminidases