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标题：Simple and rapid biochemical method to synthesize labeled or unlabeled phosphatidylinositol species
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作者：Satu Hänninen ; Krishna Chaithanya Batchu ; Kati Hokynar 等
期刊名称：JLR Papers In Press
印刷版ISSN：0022-2275
电子版ISSN：1539-7262
出版年度：2017
卷号：58
期号：6
页码：1259-1264
DOI：10.1194/jlr.D075960
语种：English
出版社：American Society for Biochemistry and Molecular Biology
摘要：Phosphatidylinositol (PI) is the precursor of many important signaling molecules in eukaryotic cells and, most probably, PI also has important functions in cellular membranes. However, these functions are poorly understood, which is largely due to that i ) only few PI species with specific acyl chains are available commercially and ii ) there are no simple methods to synthesize such species. Here, we present a simple biochemical protocol to synthesize a variety of labeled or unlabeled PI species from corresponding commercially available phosphatidylcholines. The protocol can be carried out in a single vial in a two-step process which employs three enzymatic reactions mediated by i ) commercial phospholipase D from Streptomyces chromofuscus , ii ) CDP-diacylglycerol synthase overexpressed in E. coli and iii ) PI synthase of Arabidopsis thaliana ectopically expressed in E. coli . The PI product is readily purified from the reaction mixture by liquid chromatography since E. coli does not contain endogenous PI or other coeluting lipids. The method allows one to synthesize and purify labeled or unlabeled PI species in 1 or 2 days.Typically, 40–60% of (unsaturated) PC was converted to PI albeit the final yield of PI was less (25–35%) due to losses upon purification.
关键词：phospholipids/biosynthesis ; enzymology ; molecular biology ; methods/HPLC ; mass spectrometry