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 degrees 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 microM depending on the species of lyso-form when [14C]stearic acid was used at the concentration of 100 microM. 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.
Mitsutake
S
S
Laboratory of Marine Biochemistry, Faculty of Agriculture, Kyushu University, 6-10-1, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
Kita
K
K
Nakagawa
T
T
Ito
M
M
D000581:Amidohydrolases / Q000032:analysis / Q000378:metabolism*; D000818:Animals; D002250:Carbon Radioisotopes; D002855:Chromatography, Thin Layer; D006026:Glycoside Hydrolases / Q000032:analysis / Q000378:metabolism*; D006028:Glycosphingolipids / Q000096:biosynthesis* / Q000302:isolation & purification; D006868:Hydrolysis; D012615:Sea Anemones / Q000201:enzymology*
D002250:Carbon Radioisotopes; D006028:Glycosphingolipids; D006026:Glycoside Hydrolases; C050427:endoglycoceramidase; D000581:Amidohydrolases; C096410:sphingolipid ceramide N-deacylase