ANALYSIS OF GLUCOCEREBROSIDASE ACTIVITY USING N-(1-[C-14]HEXANOYL)-D-ERYTHRO-GLUCOSYLSPHINGOSINE DEMONSTRATES A CORRELATION BETWEEN LEVELS OF RESIDUAL ENZYME-ACTIVITY AND THE TYPE OF GAUCHER DISEASE

Glucosylceramide, a degradation product of complex glycosphingolipids, is hydrolysed in lysosomes by glucocerebrosidase (GlcCerase). Mutatio ns in the human GlcCerase gene cause a reduction in GlcCerase activity and accumulation of glucosylceramide, which results in the onset of G aucher disease, the most common lysosomal storage disease. Significant clinical heterogeneity is observed in Gaucher disease, with three mai n types known, but no clear correlation has been reported between the different types and levels of residual GlcCerase activity. We now demo nstrate that a correlation exists by using a radioactive, short-acyl c hain substrate, N-(1-[C-14]hexanoyl)-D-erythro-glucosylsphingosine ([C -14]hexanoyl-GlcCer). This substrate rapidly transferred into biologic al membranes in the absence of detergent [Futerman and Pagano (1991) B iochem. J. 280, 295-302] and was hydrolyzed to N-(1-[C-14]hexanoyl)-D- erythro-sphingosine ([C-14]hexanoyl-Cer) both in vitro and in situ, wi th an acid pH optimum. A strict correlation was observed between level s of [C-14]hexanoyl-GlcCer hydrolysis and Gaucher type in human skin f ibroblasts. The mean residual activity measured in vitro for 3 h incub ation in type 1 Gaucher fibroblasts (the mild form of the disease) was 46.3 +/- 4.6 nmol of [C-14]hexanoyl-Cer formed per mg protein (n = 9) , and in type 2 and 3 fibroblasts (the neuronopathic forms of the dise ase) was 19.6 +/- 6.5 (n = 9). A similar correlation was observed when activity was measured in situ, suggesting that the clinical severity of a lysosomal storage disease is related to levels of residual enzyme activity.