Liposome-Mediated, Fluorescence-Based Studies of Sphingolipid Metabolism in Intact Cells

Our laboratory has been engaged in studies of lipid hydrolases since the early 1960s. In the course of these studies the following enzymes have been isolated and partially purified: ceramidase (1 –3 ); two sphingomyelinases: the lysosomal, with an acidic pH optimum (4 ) and a neutral, magnesiumdependent (5 -7 ) enzyme; β-glucosidase and α- or β-galactosidase (8 –11 ); N -acetyl hexosaminidase (12 –14 ); neuraminidase (15 ); phospholipase A1 (16 ,17 ), lysophospholipase (18 –20 ), and brain lipase (21 –23 ). For assaying these enzymes we labeled the respective substrates with tritium (3 H) or 14 C and developed procedures for separating the hydrolytic products from the, as yet, nondegraded substrates. In the late 1970s we began synthesizing colored and flurorescent derivatives of lipids. In 1981 in Methods in Enzymology , vol. 72 (24 –32 ), we presented a comprehensive description of the assay procedures using sphingolipids to which we linked trinitrophenol as a colored, yellow marker and the fluorescent probes anthracene, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD), pyrene, carbazole, and dansyl. More recently we replaced these with the more polar fluorescent probe, lissamine rhodamine (LR), or with Bodipy; these probes were linked to the sphingolipids via a 12-carbon spacer (LR12 and Bod12) and were used for assaying lipid hydrolases in vitro (33 –39 ). The availability of fluorescent lipids opened for us a new approach, that is, their administration into intact cells in culture and following their intracellular metabolism (40 –47 ). In most cases our interest was in their enzymatic hydrolysis within the intact lysosomes of the living cells, normal or derived from patients with lipid storage disorders (sphingolipidoses).