- Bioluminescent Assay for Sphingolipid Ceramide N-Deacylase Using Vibrio harveyi Dark Mutant M-17
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Ki Woong Cho
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J. Microbiol. 2008;46(5):585-589. Published online October 31, 2008
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DOI: https://doi.org/10.1007/s12275-008-0114-2
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A new bioluminescent assay method for the activity of sphingolipid ceramide N-deacylase (SCDase: EC3.5.1.69) as well as ceramidase (CDase: EC 3.5.1.23) was developed using bioluminescent marine bacteria. Enzymatically synthesized ceramide (N-myristoyl sphigosine, C14:0-18:1) 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 μU and 5 nM of myristic acid production were detected in less than one min.
<br>The assay worked well for the determination of Km and chromatographic fraction assay.
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Citations
Citations to this article as recorded by  - Control of metabolism and signaling of simple bioactive sphingolipids: Implications in disease
Patricia Gangoiti, Luz Camacho, Lide Arana, Alberto Ouro, Maria H. Granado, Leyre Brizuela, Josefina Casas, Gemma Fabriás, José Luis Abad, Antonio Delgado, Antonio Gómez-Muñoz
Progress in Lipid Research.2010; 49(4): 316. CrossRef
- Bioluminescent Assay of Phospholipase C Using A Luminescent Marine Mutant Bacterium Vibrio harveyi M-17
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Ki Woong Cho , SangJun Mo , Hyi-Seung Lee , Jung-Rae Rho , Jongheon Shin
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J. Microbiol. 2000;38(3):150-155.
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A bioluminescent assay method for detecting the activity of phospholipase C (PLC; phosphatidyl choline cholinephosphohydrolase, EC 3.1.4.3) was developed using bioluminescent marine bacteria. Phospholipase C from Bacillus cereus and sn-1,2-dimyristoyl phosphatidyl choline (DMPC) as a substrate were used in the demonstration, and the produced sn-1,2-dimyristoyl glycerol was further hydrolyzed with lipase from Candida cylidracea. The hydrolyzed myristic acid was quantified using a dark mutant of Vibrio harveyi (designated as M-17). The in vivo light intensity of which was stimulated specifically up to one thousand fold in the presence of myristic acid. The rates of the hydrolysis of the DMPC substrate by the phospholipase measured by the luminescence method were linear with time and the amount of enzyme added. Activity measurement conditions (at 25 C, pH 6.5, 10 min fixed time assay) were established to detect as little as 0.1 mUnit of phospholipase C and 5 nM of myristic acid production.
- Bioluminescent Assay of Bovine Liver Riboflavin Kinase Using a Bacterial Luciferase Coupled Reaction
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Ki Woong Cho
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J. Microbiol. 2000;38(2):74-79.
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For the demonstration of a novel riboflavin kinase assay method based on the bacterial bioluminescence, partially purified riboflavin kinase was prepared from bovine liver through ammonium sulfate precipitation and DEAE-cellulose ion exchange chromatography. Using bacterial luciferase from Photobacterium phosphoreum and the dithionite reduction method, an easy, safe, and fast assay method was established. The optimal temperature, pH, Km values for riboflavin and ATP of bovine liver riboflavin kinase determined with this luminescence method were 35 C, pH 7, 15.3 uM and 8.3 uM, respectively. The detection limit of FMN produced by riboflavin kinase was in the range of 200 pM to 4 uM which is comparable to the HPLC-fluorescence detection method, while the detection time for each assay was less than 15 sec compared to the HPLC method which requires at least 10 min for completion.
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