A novel enzyme that cleaves the N-acyl linkage of ceramides in various glycosphingolipids as well as sphingomyelin to produce their lyso forms.
We describe a novel enzyme that hydrolyzes the N-acyl linkage between fatty acids and sphingosine bases in ceramides of various sphingolipids. The enzyme was purified about 3000-fold with 5% recovery from the culture filtrate of a newly isolated bacterium (Pseudomonas sp. TK4) by ammonium sulfate precipitation followed by several steps of high performance liquid chromatography. The purified enzyme preparation was completely free of exoglycosidases, sphingomyelinase, and proteases, and showed a single protein band corresponding to a molecular mass of 52 kDa on SDS-polyacrylamide slab gel electrophoresis after staining with Coomassie Brilliant Blue. The enzyme shows quite wide specificity, i.e. it hydrolyzes both neutral and acidic glycosphingolipids, and simple glycosphingolipid cerebrosides to polysialogangliosides such as GQ1b. Furthermore the enzyme also hydrolyzes sphingomyelin to produce the respective lyso form. However, the enzyme shows hardly any activity on ceramides, indicating that it is completely different from the ceramidase (EC 3.5.1.23) reported previously. This enzyme, which is tentatively named sphingolipid ceramide N-deacylase, should greatly facilitate the further study of sphingolipids as well as lysosphingolipids.
Ito
M
M
Laboratory of Marine Biochemistry, Faculty of Agriculture, Kyushu University, Fukuoka, Japan.
Kurita
T
T
Kita
K
K
D000581:Amidohydrolases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001921:Brain; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D002417:Cattle; D002518:Ceramides / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006028:Glycosphingolipids / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000737:chemistry; D011549:Pseudomonas / Q000201:enzymology* / Q000254:growth & development; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013109:Sphingomyelins / Q000378:metabolism*; D013379:Substrate Specificity
D002413:Cations, Divalent; D002518:Ceramides; D006028:Glycosphingolipids; D009844:Oligosaccharides; D013109:Sphingomyelins; D000581:Amidohydrolases; C096410:sphingolipid ceramide N-deacylase