IN-SITU ASSAY OF ACID SPHINGOMYELINASE AND CERAMIDASE BASED ON LDL-MEDIATED LYSOSOMAL TARGETING OF CERAMIDE-LABELED SPHINGOMYELIN

The activity of lysosomal sphingolipid hydrolases is usually estimated in vitro from complex assays on cell lysates under artificial conditi ons including the presence of detergents and substrate analogs. Howeve r, the measure of their effective activity in situ (i.e., in living ce lls) is necessary to understand the normal intracellular sphingolipid turnover. Moreover, their determination in cells from patients with ge netic enzyme deficiencies represents a key parameter of the pathophysi ology of sphingolipid storage disorders. In this report, we have devel oped a procedure for estimating the effective activity oflysosomal sph ingomyelinase and ceramidase in situ. This procedure is based on the s elective targeting to lysosomes of a natural substrate under physiolog ical conditions of substrate influx. Epstein-Barr virus-transformed hu man lymphoid cells and human skin fibroblasts were incubated with puri fied human low density lipoproteins (LDL) containing [H-3] ceramide-la beled sphingomyelin. Data demonstrate that this substrate is internali zed through the apolipoprotein B/E receptor pathway and targeted to ly sosomes. Lysosomal localization of the incorporated substrate was evid ence by ultrastructural autoradiography and subcellular fractionation as well as by metabolic studies in mutant cells. Short-term pulse-chas e experiments with LDL-associated [H-3] ceramide-labeled sphingomyelin allowed us to determine the effective activity of lysosomal sphingomy elinase and ceramidase in normal cells. Initial Velocities of sphingom yelin and ceramide degradation were, respectively, estimated at 0.66 a nd 1.14 nmol . h(-1). mg cell protein(-1) in lymphoid cells, and 5.4 a nd 3 nmol . h(-1). mg cell protein(-1) in skin fibroblasts.