title: Preparation of fluorescence-labeled GM1 and sphingomyelin by the reverse hydrolysis reaction of sphingolipid ceramide N-deacylase as substrates for assay of sphingolipid-degrading enzymes and for detection of sphingolipid-binding proteins.
abstract: Sphingolipid ceramide N-deacylase is an enzyme capable of hydrolyzing the N-acyl linkages of ceramides of various sphingolipids. Recently, it was found that the enzyme catalyzes the reverse hydrolysis reaction in which free fatty acids are condensed to lyso-sphingolipids to produce sphingolipids. This paper describes a simple method for the synthesis of fluorescence-labeled sphingolipids utilizing the condensation reaction of the enzyme. N-TFAc-aminododecanoic acids were efficiently condensed by the enzyme to the lyso-forms of GM1 and sphingomyelin in glycine buffer (pH 10). The reaction products, N-TFAc-amino-GM1 and sphingomyelin, were obtained with overall yields of 60%. The purified products were identified to be omega-amino-GM1 and omega-amino-sphingomyelin, respectively, by TLC and FAB-MS or ESI-LC/MS analysis after removal of the N-TFAc by mild alkaline treatment. NBD-labeled GM1 and sphingomyelin were prepared from omega-amino-GM1 and omega-amino-sphingomyelin by coupling with 4-fluoro-NBD. These fluorescence-labeled substrates, C12-NBD-GM1 and C12-NBD-sphingomyelin, were hydrolyzed by endoglycoceramidase and sphingomyelinase, respectively, to produce NBD-dodecanoylsphingosines, but were resistant to hydrolysis by sphingolipid ceramide N-deacylase. C12-NBD-sphingomyelin was found to be a better substrate than the commercially available C6-NBD-sphingomyelin for the assay of sphingomyelinase from various sources. We also describe a new method to detect GM1-binding proteins using fluorescence-labeled GM1.
authors.affiliation: Department of Bioscience and Biotechnology, Division of Bioresource and Bioenvironmental Sciences, Graduate School Kyushu University, Higashi-ku, Fukuoka, 812-8581, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Nakagawa
authors.forename: M
authors.initials: M
authors.lastname: Tani
authors.forename: K
authors.initials: K
authors.lastname: Kita
authors.forename: M
authors.initials: M
authors.lastname: Ito
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000201:enzymology; D002855:Chromatography, Thin Layer; D005456:Fluorescent Dyes / Q000737:chemistry; D005677:G(M1) Ganglioside / Q000737:chemistry* / Q000378:metabolism; D006868:Hydrolysis; D007668:Kidney / Q000201:enzymology; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D011485:Protein Binding; D013109:Sphingomyelins / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D005456:Fluorescent Dyes; D013109:Sphingomyelins; D005677:G(M1) Ganglioside; D000581:Amidohydrolases; C096410:sphingolipid ceramide N-deacylase