Document Detail


Enzymatic measurement of phosphatidic acid in cultured cells.
MedLine Citation:
PMID:  19369695     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
In this work, we developed a novel enzymatic method for measuring phosphatidic acid (PA) in cultured cells. The enzymatic reaction sequence of the method involves hydrolysis of PA to produce glycerol-3-phosphate (G3P), which is then oxidized by G3P oxidase to generate hydrogen peroxide. In the presence of peroxidase, hydrogen peroxide reacted with Amplex Red to produce highly fluorescent resorufin. We found that lipase from Pseudomonas sp. can completely hydrolyze PA to G3P and FAs. The calibration curve for PA measurement was linear between 20 and 250 microM, and the detection limit was 5 microM (50 pmol in the reaction mixture). We also modified the method for the enzymatic measurement of lysophosphatidic acid. By this new method, we determined the PA content in the lipid extract from HEK293 cells. The cellular content of PA was decreased with increasing cell density but not correlated with the proliferation rate. The diacylglycerol kinase inhibitor R59949 markedly reduced the cellular PA content, suggesting the diacylglycerol kinase activity was involved in a large part of the PA production in HEK293 cells. This novel method for PA quantification is simple, rapid, specific, sensitive, and high-throughput and will help to study the biological functions of PA and its related enzymes.
Authors:
Shin-ya Morita; Kazumitsu Ueda; Shuji Kitagawa
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2009-04-15
Journal Detail:
Title:  Journal of lipid research     Volume:  50     ISSN:  0022-2275     ISO Abbreviation:  J. Lipid Res.     Publication Date:  2009 Sep 
Date Detail:
Created Date:  2009-12-04     Completed Date:  2010-02-08     Revised Date:  2013-06-02    
Medline Journal Info:
Nlm Unique ID:  0376606     Medline TA:  J Lipid Res     Country:  United States    
Other Details:
Languages:  eng     Pagination:  1945-52     Citation Subset:  IM    
Affiliation:
Laboratory of Pharmaceutical Technology, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan. smorita@kobepharma-u.ac.jp
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MeSH Terms
Descriptor/Qualifier:
Bacteria / enzymology
Cell Count
Cell Line
Diacylglycerol Kinase / antagonists & inhibitors
Enzyme Inhibitors / pharmacology
Enzymes / metabolism*
Humans
Lipids / chemistry
Lysophospholipids / analysis
Phosphatidic Acids / analysis*,  metabolism*
Time Factors
Chemical
Reg. No./Substance:
0/Enzyme Inhibitors; 0/Enzymes; 0/Lipids; 0/Lysophospholipids; 0/Phosphatidic Acids; 22002-87-5/lysophosphatidic acid; EC 2.7.1.107/Diacylglycerol Kinase
Comments/Corrections

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