Enzymatic synthesis of 14C-glycosphingolipids by reverse hydrolysis reaction of sphingolipid ceramide N-deacylase: detection of endoglycoceramidase activity in a seaflower.

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Abstract
This paper describes the synthesis of 14C-labeled glycosphingolipids using the reverse hydrolysis reaction (condensation) of sphingolipid ceramide N-deacylase. It was found that 50–70% of 14C-fatty acids were incorporated into various lyso-glycosphingolipids when a mixture of lyso-glycosphingolipids and fatty acids was incubated at 37°C with 1 mU of the enzyme for 20 h in 1 ml of 25 mM phosphate buffer, pH 6.0–7.0, containing 0–0.1% Triton X-100. The optimum concentration of lyso-glycosphingolipids was 100–400 μM depending on the species of lyso-form when [14C] stearic acid was used at the concentration of 100 μM. Free 14C-fatty acids and lyso-glycosphingolipids were separated from the synthesized 14C-glycosphingolipids by using a Sep-Pak Plus Silica and a Sep-Pak CM or a QMA cartridge, respectively. After treatment of 14C-glycosphingolipids with endoglycoceramidase or sphingolipid ceramide N-deacylase, digestion products were clearly separated from the parent glycosphingolipids on TLC and determined using an image analyzer with a sensitivity 100 times higher than that using non-radiolabeled substrates. Using this method, we found endoglycoceramidase activity in a seaflower, Condylactis sp., for the first time.

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