Ganglioside GM2 N-acetyl-beta-D-galactosaminidase and asialo GM2 (GA2) N-acetyl-beta-D-galactosaminidase; studies in human skin fibroblasts.

Abstract

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.

• Authors: J S O'Brien, G W Norden, A L Miller, R G Frost, T E Kelly
• Journal: Clinical genetics (Clin Genet) Vol. 11 Issue 3 Pg. 171-83 (Mar 1977) ISSN: 0009-9163 [Print] Denmark
• PMID: 13950 (Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Albumins
• Borohydrides
• Detergents
• Gangliosides
• Lipids
• G(M2) Ganglioside
• Taurocholic Acid
• Galactose Oxidase
• Hexosaminidases

Topics

• Adult
• Albumins (pharmacology)
• Borohydrides (pharmacology)
• Cells, Cultured
• Chromatography, Thin Layer
• Detergents (pharmacology)
• Female
• Fibroblasts (enzymology)
• G(M2) Ganglioside (analogs & derivatives, metabolism)
• Galactose Oxidase (pharmacology)
• Gangliosides (metabolism)
• Gangliosidoses (enzymology)
• Hexosaminidases (deficiency, metabolism)
• Humans
• Hydrogen-Ion Concentration
• Isotope Labeling
• Lipidoses (enzymology)
• Lipids (pharmacology)
• Skin (enzymology)
• Taurocholic Acid (pharmacology)