A novel assay method for glycosphingolipid deacylase by enzyme-linked immunochemical detection of lysoglycosphingolipid.
Lysoglycosphingolipids consist of a sphingoid long-chain base and monosaccharide or complex sugar, and they lack the fatty acyl group present in native glycosphingolipids. Less than 1 pmol of lyso-Forssman glycolipid and lysoganglioside GM1 were detected on a thin-layer chromatogram by an enzyme-linked immunochemical coloration method with anti-Forssman glycolipid antibody (FOM-1) and cholera toxin B subunit, respectively. Each spot between 1 and 100 pmol lyso-Forssman glycolipid was immunostained as densely as that of the same amount of native Forssman glycolipid. The density of the lyso-Forssman glycolipid spots increased proportionally with increment in the amount of lysoglycolipid. The density of spots of 0.2-100 pmol lysoganglioside GM1 was also proportional to the amount of each lyso-GM1 spot. These results indicated that less than 1 to 100 pmol of deacylated glycosphingolipid was quantifiable by the immunochemical coloration method with sugar chain-specific antibodies. Glycosphingolipid deacylase, which cleaved an amide bond between the sphingoid long-chain base and fatty acyl chain in ceramide of glycosphingolipid, was assayed by detecting the lyso-Forssman glycolipid produced. Lipophilic compounds, recovered from an aliquot of the reaction mixture of Forssman glycolipid and crude enzyme at appropriate times, were analyzed by thin-layer chromatography. It was found that lyso-Forssman glycolipid was produced in the first 1-2 h by the enzyme and production increased with incubation time. This coloration method is more sensitive and specific than the visualization method with a nonspecific reagent such as orcinol-sulfuric acid reagent.
Izumi
K
K
Hokkaido National Industrial Research Institute, AIST, MITI, Sapporo, Japan. izumi@hniri.go.jp
Sawada
M T
MT
D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000032:analysis* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D005677:G(M1) Ganglioside / Q000031:analogs & derivatives* / Q000032:analysis* / Q000378:metabolism; D005915:Globosides / Q000032:analysis / Q000378:metabolism; D006017:Glycolipids / Q000032:analysis* / Q000378:metabolism; D006736:Horses; D007124:Immunoenzyme Techniques; D012240:Rhodococcus / Q000201:enzymology; D013552:Swine
D005915:Globosides; D006017:Glycolipids; C029465:lysoGM1 ganglioside; D005677:G(M1) Ganglioside; C010163:Forssman glycolipid; D002265:Carboxylic Ester Hydrolases; C422848:glycosphingolipid deacylase