CLASSIFICATION OF DISORDERS OF G(M2) GANGLIOSIDE HYDROLYSIS USING H-3G(M2) AS SUBSTRATE

Rates of G(M2) ganglioside hydrolysis by fibroblasts from normal contr ols and patients with G(M2) gangliosidosis were measured in situ, with cells growing in tissue culture by assaying the decrease in cell-inco rporated H-3-G(M2) over time, and in vitro by assaying the rate of H-3 -G(M2) hydrolysis using fibroblast extracts in the presence of no addi tives, sodium taurocholate, and G(M2) activator protein. In tissue cul ture, normal cells hydrolyzed cell-incorporated G(M2) while fibroblast s from patients with G(M2) gangliosidosis did not. The half life of G( M2) in normal fibroblasts was 78 hours. In vitro, only normal fibrobla st extracts hydrolyzed G(M2) in the absence of additives. In the prese nce 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 ext racts from patients with Tay-Sachs disease hydrolyzed G(M2) at nearly normal rates, and cell extracts from Sandhoff disease patients hydroly zed G(M2) at about 10% of normal rates. In the presence of 1 mu g of G (M2) activator, rates of G(M2) hydrolysis by normal fibroblast extract s were increased 8-25-fold, fibroblast extracts from a patient with th e AB variant hydrolyzed G(M2) at normal rates, and cell extracts from other variants of G(M2) gangliosidosis did not hydrolyze G(M2). The re sults suggest that measuring the persistence of H-3-G(M2) in tissue cu lture over time will detect any variant of G(M2) gangliosidosis and ma y be the ideal way to test for the presence of this disease. Variants can be distinguished by assaying the hydrolysis of H-3-G(M2) using cel l extracts in the absence of additives, with sodium taurocholate, and with activator.