Characterization of human acid sphingomyelinase purified from the media of overexpressing Chinese hamster ovary cells.
A rapid purification method was developed to isolate milligram quantities of human acid sphingomyelinase from the media of overexpressing Chinese hamster ovary cells. The purified, recombinant enzyme (rhASM) had physical and kinetic characteristics that were consistent with those reported for the non-recombinant enzyme, including an acidic pH optimum and sensitivity to sulfhydryl reducing reagents and the zinc specific chelator, 1, 10-phenanthroline. A novel assay using fluorescently conjugated sphingomyelin was developed to explore the substrate binding properties of rhASM. Substrate binding required a fatty acid chain length of at least six carbons and the presence of the phosphocholine headgroup on sphingomyelin. Substrate binding also required an acidic pH, and was inhibited by pretreatment of the enzyme with sulfhydral reducing reagents or 1,10-phenanthroline. rhASM was rapidly internalized by cultured skin fibroblasts from Niemann-Pick disease (NPD) patients, and approximately 50% of this uptake was dependent on the mannose 6-phosphate receptor system. Studies using FITC-labeled rhASM revealed that by 1 h the internalized enzyme was localized to acidic compartments and could degrade sphingomyelin, the first demonstration that a lysosomal sphingolipid hydrolase can be fluorescently labeled and retain its biological activity. Intravenous injection of rhASM into ASM knock-out mice showed that the t(1/2) in the plasma was less than 5 min, and that the majority of the injected enzyme was taken up by the liver, followed by the spleen. Thus, these studies lay the foundation for future structure/function investigations of ASM, further investigations into this enzyme's role in ceramide mediated signal transduction, and the evaluation of enzyme replacement therapy for NPD using the mouse model.
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Department of Human Genetics, Box 1498, Mount Sinai School of Medicine, 1425 Madison Avenue, Room 14-20A, New York, NY 10029, USA.
Miranda
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Xiong
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Dagan
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Gatt
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Schuchman
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D000818:Animals; D016466:CHO Cells / Q000201:enzymology*; D006224:Cricetinae; D003470:Culture Media / Q000737:chemistry; D004229:Dithiothreitol / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D009542:Niemann-Pick Diseases / Q000378:metabolism; D010618:Phenanthrolines / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry; D013108:Sphingomyelin Phosphodiesterase / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D014018:Tissue Distribution
D003470:Culture Media; D010618:Phenanthrolines; D011994:Recombinant Proteins; C112980:acid sphingomyelinase-1; D013108:Sphingomyelin Phosphodiesterase; D016650:Fluorescein-5-isothiocyanate; D004229:Dithiothreitol; C025205:1,10-phenanthroline