Bioluminescent assay for sphingolipid ceramide N-deacylase using Vibrio harveyi dark mutant M-17.
A new bioluminescent assay method for the activity of sphingolipid ceramide N-deacylase (SCDase: EC 3.5.1.69) as well as ceramidase (CDase: EC 3.5.1.23) was developed using bioluminescent marine bacteria. Enzymatically synthesized ceramide (N-myristoyl sphigosine, C14:0-18:l) and commercial SCDase were used in this demonstration, and myristic (tetradecanoic, C14:0) acid produced by the SCDase hydrolysis was quantified using Vibrio harveyi M-17, a dark mutant of V. harveyi. The in vivo light intensity of M-17 was stimulated up to thousands fold in the presence of myristic acid, was used for this assay. SCDase activity with as little as 10 microU and 5 nM of myristic acid production were detected in less than one min. The assay worked well for the determination of Km and chromatographic fraction assay.
Cho
Ki Woong
KW
Department of Marine Biotechnology, Anyang University, Incheon 417-833, Republic of Korea. kwcho@anyang.ac.kr
D000581:Amidohydrolases / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D002518:Ceramides / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods*; D009154:Mutation; D012680:Sensitivity and Specificity; D014733:Vibrio / Q000201:enzymology*
D001426:Bacterial Proteins; D002518:Ceramides; D000581:Amidohydrolases; C096410:sphingolipid ceramide N-deacylase