Intracellular degradation of fluorescent glycolipids by lysosomal enzymes and their activators

Fluorescent glycolipids were utilized for detection of the intracellular, a ctivator-dependent, activities of beta-glucocerebrosidase and arylsulphatas e A. Activities were measured in primary skin fibroblasts from normal indiv iduals, from patients with Gaucher disease who had mutations within the bet a-glucocerebrosidase gene, and from a prosaposin-deficient patient. Fluores cent microscopy demonstrated that glucosylceramide or sulphatide labelled w ith a fluorescent probe (lissamine-rhodamine) were endocytosed and reached the lysosomes. There, in the presence of active enzyme and the correspondin g saposin, they were hydrolysed to fluorescent ceramide, which changed its intracellular localization. When these substrates were labelled with pH-sen sitive lissamine-rhodamine, which loses its fluorescence at neutral or alka line pH, the transport of the product, i.e. fluorescent ceramide, from the lysosomes resulted in disappearance of the cellular fluorescence. In cells of patients having mutations within the genes encoding the glucocerebrosida se or the prosaposin, there was a considerable reduction in the intracellul ar rate of substrate hydrolysis that could be followed by fluorescence micr oscopy or measured quantitatively in cell extracts.