title: Assay to measure sphingomyelinase and ceramidase activities efficiently and safely.
abstract: As part of the sphingomyelin pathway, sphingomyelinases and ceramidases have attracted much attention in basic as well as clinical research. However, current assays still often rely on a radioactive substrate, extensive manual purification steps, and hazardous solvents for chromatographic analysis. We here show the equivalence of a fluorescent sphingomyelin substrate and present a new versatile solvent replacing the chloroform/methanol mixture. By further modifications including the omission of the manual extraction steps, chloroform and methanol are eliminated from the entire procedure and render the assay flexible to repeated analyses at multiple time intervals. These improvements allow for the rapid detection of both enzymes in a high throughput microtiter format. Moreover, we demonstrate the relevance of the plastic assay material and the interchangeability between serum and different plasma sources.
authors.affiliation: Department of Psychiatry and Psychotherapy, Friedrich-Alexander University of Erlangen-Nuremberg (FAU), Schwabachanlage 6, D-91054, Germany. Electronic address: christiane.muehle@uk-erlangen.de.
authors.forename: Christiane
authors.initials: C
authors.lastname: Mühle
authors.affiliation: Department of Psychiatry and Psychotherapy, Friedrich-Alexander University of Erlangen-Nuremberg (FAU), Schwabachanlage 6, D-91054, Germany. Electronic address: johannes.kornhuber@uk-erlangen.de.
authors.forename: Johannes
authors.initials: J
authors.lastname: Kornhuber
mesh_terms: D000085:Acetates / Q000737:chemistry; D019342:Acetic Acid / Q000737:chemistry; D000818:Animals; D000925:Anticoagulants / Q000737:chemistry; D002250:Carbon Radioisotopes; D055572:Ceramidases / Q000378:metabolism*; D002518:Ceramides / Q000378:metabolism; D002855:Chromatography, Thin Layer; D004492:Edetic Acid / Q000737:chemistry; D057075:Enzyme Assays / Q000379:methods*; D005227:Fatty Acids / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D006493:Heparin / Q000737:chemistry; D006801:Humans; D008094:Lithium / Q000737:chemistry; D051379:Mice; D041681:NIH 3T3 Cells; D011126:Polypropylenes / Q000737:chemistry; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D044967:Serum / Q000378:metabolism; D012997:Solvents / Q000737:chemistry; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*; D013109:Sphingomyelins / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D000085:Acetates; D000925:Anticoagulants; D002250:Carbon Radioisotopes; D002518:Ceramides; D005227:Fatty Acids; D005456:Fluorescent Dyes; D011126:Polypropylenes; D011994:Recombinant Proteins; D012997:Solvents; D013109:Sphingomyelins; C007650:ethyl acetate; D006493:Heparin; D008094:Lithium; D004492:Edetic Acid; D013108:Sphingomyelin Phosphodiesterase; D055572:Ceramidases; D019342:Acetic Acid
keywords: Ceramidase; Enzyme activity assay; Fluorescent substrate; High throughput; Sphingomyelinase; Thin layer chromatography