Ganglioside GM2 N‐acetyl‐ß‐D‐gafactosaminidase and asialo GM2 (GA2) N‐acetyl‐ß‐D‐galactosaminidase; studies in human skin fibroblasts

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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 × 104 dpm/nmol (GM2) and 1.8 × 106 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‐β‐D‐galactosaminidase (E.C.3.2.1.30) and GA2‐N‐acetyl‐β‐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‐β‐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‐β‐D‐galactosaminidase and 4700 for 4‐methylumbelliferyl‐N‐acetyl‐β‐D‐glucosaminidase. Supernates from two patients with Tay‐Sachs disease had markedly reduced activity levels for GM2‐N‐acetyl‐β‐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‐β‐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.