Classification of disorders of G M2 ganglioside hydrolysis using 3H-G M2 as substrate

Abstract

Rates of G M2 ganglioside hydrolysis by fibroblasts from normal controls and patients with G M2 gangliosidosis were measured in situ, with cells growing in tissue culture by assaying the decreases in cell-incorporated 3H-G M2 over time, and in vitro by assaying the rate of 3H-G M2 hydrolysis using fibroblast extracts in the presence of no additives, spdium taurocholate, and G M2 activator protein. In tissue culture, normal cells hydrolyzed cell-incorporated G M2 while fibroblasts from patients with G M2 gangliosidosis did not. The half life of G M2 in normal fibroblasts was 78 hours. In vitro, only normal fibroblast extracts hydrolyzed G M2 in the absence of additives. In the presence of 10 mM sodium taurocholate, rates of G M2 hydrolysis by normal fibroblast extracts were increased 5–16 fold, fibroblast extracts from AB and B1 variant patients hydrolyzed G M2 at normal rates, cell extracts from patients with Tay-Sachs disease hydrolyzed G M2 at nearly normal rates, and cell extracts from Sandhoff disease patients hydrolyzed G M2 at about 10% of normal rates. In the presence of 1 μg of G M2 activator activator, rates of G M2 hydrolysis by normal fibroblast extracts were increased 8–25-fold, fibroblast extracts form a patient with the AB variant hydrolyzed G M2. The results suggest that measuring the persistence of 3H-G M2 in tissure culture over time will detect any variant of G M2 gangliosidosis and may be the ideal way to test for the presence of this disease. Variants can be distinguished by assaying the hydrolysis of 3H-G M2 using cell extracts in the absence of additives, with sodium taurocholate, and with activator.