Abstract
We synthesized fluorescent derivatives of sphingomyelin (N-acyl-sphingosylphosphocholine) and used them as substrates for several sphingomyelinases. The following five fluorescent probes, each attached to the terminal carbon atom of the fatty acyl residue, were introduced into sphingomyelin: dansyl, pyrene, carbazole, 4-chloro-7-nitrobenz-2-oxa-1,3-diazole, and anthroic acid. We compared the rates at which the fluoro- and radiolabeled sphingomyelins were hydrolyzed. They were the same with the following three spingomyelinases: (a) a purified enzyme from Staphylococcus aureus; (b) at Triton X100-treated extract of human brain (assayed at pH 7.4 in the presence of Mg2+; and (c) aqueous extracts of brain lysosomes, skin fibroblasts, and amniotic cells, assayed at pH 5.0. Homogenates of skin fibroblasts of a patient with Niemann-Pick disease had practically no activity when assayed at pH 5, with fluorosphingomyelin as substrate. When fluorosphingomyelin was mixed in various proportions with natural sphingomyelin, enzymes from each of the three sources hydrolyzed the two substrates at equal rates. The fluorosphingomyelins can be used to estimate with great sensitivity the sphingomyelinase activity in extracts of tissues or cells, in tears, and probably in hair follicles, as well as diagnose Niemann-Pick disease, either pre- or post-natally
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