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

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.