Classification of disorders of GM2 ganglioside hydrolysis using 3H-GM2 as substrate.

Rates of GM2 ganglioside hydrolysis by fibroblasts from normal controls and patients with GM2 gangliosidosis were measured in situ, with cells growing in tissue culture by assaying the decrease in cell-incorporated 3H-GM2 over time, and in vitro by assaying the rate of 3H-GM2 hydrolysis using fibroblast extracts in the presence of no additives, sodium taurocholate, and GM2 activator protein. In tissue culture, normal cells hydrolyzed cell-incorporated GM2 while fibroblasts from patients with GM2 gangliosidosis did not. The half life of GM2 in normal fibroblasts was 78 hours. In vitro, only normal fibroblast extracts hydrolyzed GM2 in the absence of additives. In the presence of 10 mM sodium taurocholate, rates of GM2 hydrolysis by normal fibroblast extracts were increased 5-16-fold, fibroblast extracts from AB and B1 variant patients hydrolyzed GM2 at normal rates, cell extracts from patients with Tay-Sachs disease hydrolyzed GM2 at nearly normal rates, and cell extracts from Sandhoff disease patients hydrolyzed GM2 at about 10% of normal rates. In the presence of 1 microgram of GM2 activator, rates of GM2 hydrolysis by normal fibroblast extracts were increased 8-25-fold, fibroblast extracts from a patient with the AB variant hydrolyzed GM2 at normal rates, and cell extracts from other variants of GM2 gangliosidosis did not hydrolyze GM2. The results suggest that measuring the persistence of 3H-GM2 in tissue culture over time will detect any variant of GM2 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-GM2 using cell extracts in the absence of additives, with sodium taurocholate, and with activator.