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 conditions including the presence of detergents and substrate analogs. However, the measure of their effective activity in situ (i.e., in living cells) is necessary to understand the normal intracellular sphingolipid turnover. Moreover, their determination in cells from patients with genetic enzyme deficiencies represents a key parameter of the pathophysiology of sphingolipid storage disorders. In this report, we have developed a procedure for estimating the effective activity of lysosomal sphingomyelinase and ceramidase in situ. This procedure is based on the selective targeting to lysosomes of a natural substrate under physiological conditions of substrate influx. Epstein-Barr virus-transformed human lymphoid cells and human skin fibroblasts were incubated with purified human low density lipoproteins (LDL) containing [3H]ceramide-labeled sphingomyelin. Data demonstrate that this substrate is internalized through the apolipoprotein B/E receptor pathway and targeted to lysosomes. Lysosomal localization of the incorporated substrate was evidenced by ultrastructural autoradiography and subcellular fractionation as well as by metabolic studies in mutant cells. Short-term pulse-chase experiments with LDL-associated [3H]ceramide-labeled sphingomyelin allowed us to determine the effective activity of lysosomal sphingomyelinase and ceramidase in normal cells. Initial velocities of sphingomyelin and ceramide degradation were, respectively, estimated at 0.66 and 1.14 nmol.h-1.mg cell protein-1 in lymphoid cells, and 5.4 and 3 nmol.h-1.mg cell protein-1 in skin fibroblasts. The advantages and applications of these in situ studies are discussed.
Levade
T
T
Laboratoire de Biochimie, CJF INSERM 9206, Institut Louis Bugnard, C.H.U. Rangueil, Toulouse, France.
Leruth
M
M
Graber
D
D
Moisand
A
A
Vermeersch
S
S
Salvayre
R
R
Courtoy
P J
PJ
D000581:Amidohydrolases / Q000032:analysis*; D001345:Autoradiography; D001681:Biological Assay; D002460:Cell Line; D055572:Ceramidases; D002518:Ceramides; D006801:Humans; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism* / Q000648:ultrastructure; D008854:Microscopy, Electron; D013108:Sphingomyelin Phosphodiesterase / Q000032:analysis*; D013109:Sphingomyelins / Q000378:metabolism*; D014316:Tritium
D002518:Ceramides; D008077:Lipoproteins, LDL; D013109:Sphingomyelins; D014316:Tritium; D013108:Sphingomyelin Phosphodiesterase; D000581:Amidohydrolases; D055572:Ceramidases