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Publisher Summary This chapter focuses on the process of obtaining human acid sphingomyelinase from human urine. The purified enzyme has a low hydrolytic activity if the assay contains pure sphingomyelin liposomes and the addition of detergents stimulates activity. The incubation mixture contains 100 μM [ choline-methyl - 3 H]sphingomyelin from bovine brain, 200 mM sodium/acetate buffer (pH 4.5); 0.05% (w/v) Nonidet P-40 (NP-40) as detergent; and an aliquot of the enzyme preparation in 10 mM Tris-HCl buffer (pH 7.2), 0.1% (w/w) Nonidet P-40 or crude enzyme solution up to a volume of 20 μA. [ 3 H]Sphingomyelin dissolved in 20 μl toluene/ethanol (2 : 1, v/v) or in another organic solvent, is added to each test tube and the solvent is evaporated in a stream of nitrogen at room temperature. After adding the buffer and detergent, the solution is mixed and then cooled to 0° on ice, after which the enzyme is added. The purification steps are carried out at 4° unless otherwise stated. For successful enzyme purification, only urine with slightly elevated protein content but a highly elevated level of sphingomyelinase activity should be used. The clear supernatant from stored urine is concentrated by ultrafiltration and the supernatant is then loaded onto an octyl-Sepharose column previously equilibrated with buffer A. Unbound proteins are removed with at least 800 ml of buffer A. The octyl-Sepharose eluate containing the enzyme is loaded onto a concanavalin A- Sepharose column (1.6 × 24 cm) equilibrated with 10 mM Tris-HCl buffer (pH 7.2), 100 mM NaCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 1 mM MnCl 2 , 0.01%, NAN 3 , and 0.1% Nonidet P-40 (buffer B).

The GM2 activator protein is a glycolipid-binding protein required for the lysosomal degradation of ganglioside GM2. A human fibroblast cDNA library was screened with mixtures of oligonucleotide probes corresponding to four different areas of the amino acid sequence. A putative clone (821 bp) which gave positive signals to all four probe mixtures was purified and sequenced. The sequence was colinear with the sequence of 160 amino acids of the mature GM2 activator protein. Availability of the cDNA clone should facilitate investigation into function of the GM2 activator protein and also into genetic abnormalities underlying GM2 gangliosidosis AB variant.