A novel assay method for glycosphingolipid deacylase by enzyme-linked immunochemical detection of lysoglycosphingolipid |
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Authors: | Kazuo Izumi Michiko Takagi Sawada |
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Affiliation: | (1) Hokkaido National Industrial Research Institute. AIST. MITI, 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, 062-8517 Sapporo, Japan |
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Abstract: | 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 non-specific reagent such as orcinol-sulfuric acid reagent. |
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