DOI: https://doi.org/10.1016/j.ab.2017.03.010
書誌情報 : Anal Biochem. 2017 Jun 1;526:43-49
DOI: https://doi.org/10.1016/j.ab.2017.03.010
Development of a liquid chromatography-mass spectrometry based enzyme activity assay for phosphatidylcholine-specific phospholipase C
##article.authors##
-
Murakami, Chiaki
Aichi Cancer Center
https://orcid.org/0000-0003-3645-8382
https://researchmap.jp/murakami_chiaki
- Satoru Mizuno Chiba University
- Sayaka Kado Chiba University
- Fumio Sakane Chiba University
DOI:
https://doi.org/10.51094/jxiv.4390キーワード:
液体クロマトグラフィー-質量分析法、 ジアシルグリセロール、 ホスホリパーゼC、 酵素活性測定抄録
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.
利益相反に関する開示
開示べきCOIはありませんダウンロード *前日までの集計結果を表示します
引用文献
Y. Nishizuka, Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C, Science, 258 (1992) 607-614.
A.M. Valverde, J. Sinnett-Smith, J. Van Lint, E. Rozengurt, Molecular cloning and characterization of protein kinase D: a target for diacylglycerol and phorbol esters with a distinctive catalytic domain, Proc Natl Acad Sci U S A, 91 (1994) 8572-8576.
J.O. Ebinu, D.A. Bottorff, E.Y. Chan, S.L. Stang, R.J. Dunn, J.C. Stone, RasGRP, a Ras guanyl nucleotide- releasing protein with calcium- and diacylglycerol-binding motifs, Science, 280 (1998) 1082-1086.
M.J. Caloca, N. Fernandez, N.E. Lewin, D. Ching, R. Modali, P.M. Blumberg, M.G. Kazanietz, Beta2-chimaerin is a high affinity receptor for the phorbol ester tumor promoters, The Journal of biological chemistry, 272 (1997) 26488-26496.
N. Brose, C. Rosenmund, Move over protein kinase C, you've got company: alternative cellular effectors of diacylglycerol and phorbol esters, J Cell Sci, 115 (2002) 4399-4411.
B. Li, H. Li, Z. Wang, Y. Wang, A. Gao, Y. Cui, Y. Liu, G. Chen, Evidence for the role of phosphatidylcholine-specific phospholipase in experimental subarachnoid hemorrhage in rats, Exp Neurol, 272 (2015) 145-151.
H. Li, L. Zhang, D. Yin, Y. Zhang, J. Miao, Targeting phosphatidylcholine-specific phospholipase C for atherogenesis therapy, Trends Cardiovasc Med, 20 (2010) 172-176.
F. Podo, L. Paris, S. Cecchetti, F. Spadaro, L. Abalsamo, C. Ramoni, A. Ricci, M.E. Pisanu, F. Sardanelli, R. Canese, E. Iorio, Activation of Phosphatidylcholine-Specific Phospholipase C in Breast and Ovarian Cancer: Impact on MRS-Detected Choline Metabolic Profile and Perspectives for Targeted Therapy, Front Oncol, 6 (2016) 171.
X. Wu, H. Lu, L. Zhou, Y. Huang, H. Chen, Changes of phosphatidylcholine-specific phospholipase C in hepatocarcinogenesis and in the proliferation and differentiation of rat liver cancer cells, Cell Biol Int, 21 (1997) 375-381.
K. Goto, T. Nakano, Y. Hozumi, Diacylglycerol kinase and animal models: the pathophysiological roles in the brain and heart, Advances in enzyme regulation, 46 (2006) 192-202.
F. Sakane, S. Imai, M. Kai, S. Yasuda, H. Kanoh, Diacylglycerol kinases: why so many of them?, Biochimica et biophysica acta, 1771 (2007) 793-806.
I. Merida, A. Avila-Flores, E. Merino, Diacylglycerol kinases: at the hub of cell signalling, The Biochemical journal, 409 (2008) 1-18.
M.K. Topham, R.M. Epand, Mammalian diacylglycerol kinases: molecular interactions and biological functions of selected isoforms, Biochim Biophys Acta, 1790 (2009) 416-424.
H. Sakai, S. Kado, A. Taketomi, F. Sakane, Diacylglycerol kinase δ phosphorylates phosphatidylcholine-specific phospholipase C-dependent, palmitic acid-containing diacylglycerol species in response to high glucose levels, J Biol Chem, 289 (2014) 26607-26617.
T. Johansen, T. Holm, P.H. Guddal, K. Sletten, F.B. Haugli, C. Little, Cloning and sequencing of the gene encoding the phosphatidylcholine-preferring phospholipase C of Bacillus cereus, Gene, 65 (1988) 293-304.
I. Preuss, I. Kaiser, U. Gehring, Molecular characterization of a phosphatidylcholine-hydrolyzing phospholipase C, Eur J Biochem, 268 (2001) 5081-5091.
J.Y. Tso, C. Siebel, Cloning and expression of the phospholipase C gene from Clostridium perfringens and Clostridium bifermentans, Infect Immun, 57 (1989) 468-476.
K.Y. Hostetler, L.B. Hall, Phospholipase C activity of rat tissues, Biochem Biophys Res Commun, 96 (1980) 388-393.
M.A. Clark, R.G. Shorr, J.S. Bomalaski, Antibodies prepared to Bacillus cereus phospholipase C crossreact with a phosphatidylcholine preferring phospholipase C in mammalian cells, Biochem Biophys Res Commun, 140 (1986) 114-119.
X. He, F. Chen, M.M. McGovern, E.H. Schuchman, A fluorescence-based, high-throughput sphingomyelin assay for the analysis of Niemann-Pick disease and other disorders of sphingomyelin metabolism, Analytical biochemistry, 306 (2002) 115-123.
E.G. BLIGH, W.J. DYER, A rapid method of total lipid extraction and purification, Can J Biochem Physiol, 37 (1959) 911-917.
C.W. Cotman, Isolation of synaptosomal and synaptic plasma membrane fractions, Methods Enzymol, 31 (1974) 445-452.
F. Fonnum, Choline acetyltransferase binding to and release from membranes, Biochem J, 109 (1968) 389-398.
J.H. Zhang, T.D. Chung, K.R. Oldenburg, A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays, J Biomol Screen, 4 (1999) 67-73.
H.L. Callender, J.S. Forrester, P. Ivanova, A. Preininger, S. Milne, H.A. Brown, Quantification of diacylglycerol species from cellular extracts by electrospray ionization mass spectrometry using a linear regression algorithm, Analytical chemistry, 79 (2007) 263-272.
M. Koivusalo, P. Haimi, L. Heikinheimo, R. Kostiainen, P. Somerharju, Quantitative determination of phospholipid compositions by ESI-MS: effects of acyl chain length, unsaturation, and lipid concentration on instrument response, J Lipid Res, 42 (2001) 663-672.
C. Ramoni, F. Spadaro, B. Barletta, M.L. Dupuis, F. Podo, Phosphatidylcholine-specific phospholipase C in mitogen-stimulated fibroblasts, Exp Cell Res, 299 (2004) 370-382.
S. Cecchetti, F. Spadaro, L. Lugini, F. Podo, C. Ramoni, Functional role of phosphatidylcholine-specific phospholipase C in regulating CD16 membrane expression in natural killer cells, Eur J Immunol, 37 (2007) 2912-2922.
C. Luberto, Y.A. Hannun, Sphingomyelin synthase, a potential regulator of intracellular levels of ceramide and diacylglycerol during SV40 transformation. Does sphingomyelin synthase account for the putative phosphatidylcholine-specific phospholipase C?, J Biol Chem, 273 (1998) 14550-14559.
ダウンロード
公開済
投稿日時: 2026-05-02 02:39:01 UTC
公開日時: 2026-06-25 01:13:22 UTC
ライセンス
Copyright（c）2026
Murakami, Chiaki
Satoru Mizuno
Sayaka Kado
Fumio Sakane
この作品は、Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International Licenseの下でライセンスされています。
