Simple and rapid biochemical method to synthesize labeled or unlabeled phosphatidylinositol species

Abstract

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 ) only few PI species with specific acyl chains are available commercially and) 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 ) commercial phospholipase D from, ) CDP-diacylglycerol synthase overexpressed in and ) PI synthase of ectopically expressed in The PI product is readily purified from the reaction mixture by liquid chromatography since 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.

GhPIPLC2D promotes cotton fiber elongation by enhancing ethylene biosynthesis

“…Phosphatidylinositol (PI), the precursor of IP 3 , is composed of 1,2-DAG phosphate and inositol. As the major phospholipid in cell membranes, PI plays critical roles in various physiological processes in plants (Ha ¨nninen et al, 2017;Heilmann, 2016). Phosphorylation of PI produces phosphatidylinositol 4-phosphate (PIP 4 ) of which can be further catalyzed to generate PIP 2 (Munnik and Nielsen, 2011), a kind of membrane phospholipid involved in various developmental stages in plants (Shimada et al, 2019;Kusano et al, 2008).…”

A Novel Process Maintaining Glycerophospholipid Homeostasis in Mammalian Cells

“…The primers were obtained from Metabion International AG. Phospholipid were purchased from Avanti Polar Lipids (Alabaster, AL) or were synthesized as previously (46,47) and their purities were determined with thin layer chromatography and confirmed by mass spectrometry. Phospholipid concentrations were determined using a phosphate assay (48).…”