Development of a liquid chromatography-mass spectrometry based enzyme activity assay for phosphatidylcholine-specific phospholipase C.
Phosphatidylcholine (PC)-specific phospholipase C (PC-PLC) hydrolyzes PC to generate the second messenger 1,2-diacylglycerol (DG) and phosphocholine. PC-PLC plays pivotal roles in inflammation, carcinogenesis, tumor progression, atherogenesis, and subarachnoid hemorrhage. Although the activity of PC-PLC in mammalian tissues was discovered approximately 40 years ago, neither the protein nor its gene has been identified. In the present study, we developed a non-radioactive enzyme activity assay for PC-PLC based on mass spectrometric detection of DG following HPLC separation. This new liquid chromatography-mass spectrometry (LC-MS) assay directly determines a specific reaction product, 1-palmitoyl-2-oleoyl-DG, that is generated from 1-palmitoyl-2-oleoyl-PC by purified Bacillus cereus PC-PLC. The LC-MS assay offers several advantages including a lower background (0.02% versus 91%), higher signal background ratio (4242 versus 1.06)/signal noise ratio (7494 versus 4.4), higher sensitivity (≥32-fold), and lower limit of quantitation (0.04 pmol versus 0.69 pmol of PC-PLC), than a conventional fluorometric assay, which indirectly detects phosphocholine produced in the reaction. In addition to Bacillus cereus PC-PLC, the LC-MS assay was applicable to the measurement of mammalian PC-PLC prepared from the mouse brain. The radioisotope-free, highly sensitive and precise LC-MS assay for PC-PLC would be useful for the purification and identification of PC-PLC protein.
Department of Chemistry, Graduate School of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan.
Chiaki
C
Murakami
Department of Chemistry, Graduate School of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan.
Satoru
S
Mizuno
Center for Analytical Instrumentation, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan.
Sayaka
S
Kado
Department of Chemistry, Graduate School of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan. Electronic address: sakane@faculty.chiba-u.jp.
Fumio
F
Sakane
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002853:Chromatography, Liquid / Q000379:methods*; D004075:Diglycerides / Q000378:metabolism; D004789:Enzyme Activation; D057075:Enzyme Assays / Q000379:methods*; D006868:Hydrolysis; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D010713:Phosphatidylcholines / Q000378:metabolism; D015398:Signal Transduction; D013574:Synaptosomes / Q000201:enzymology*; D010738:Type C Phospholipases / Q000378:metabolism*
C011439:1,2-diacylglycerol; D004075:Diglycerides; D010713:Phosphatidylcholines; D010738:Type C Phospholipases; C105598:phosphatidylcholine-specific phospholipase C; C028694:1-palmitoyl-2-oleoylphosphatidylcholine
Diacylglycerol; Liquid chromatography-mass spectrometry; Phosphatidylcholine; Phosphatidylcholine-specific phospholipase C