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Simple and rapid biochemical method to synthesize labeled or unlabeled phosphatidylinositol species.Journal of Lipid Research Jun 2017Phosphatidylinositol (PI) is the precursor of many important signaling molecules in eukaryotic cells and, most probably, PI also has important functions in cellular...
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.
Topics: Biocatalysis; Chemistry Techniques, Synthetic; Isotope Labeling; Kinetics; Phosphatidylcholines; Phosphatidylinositols
PubMed: 28420658
DOI: 10.1194/jlr.D075960 -
Proceedings of the National Academy of... Oct 1991We have synthesized phosphatidylinositol 3-phosphate from phosphatidylinositol 4-phosphate by using diisopropylcarbodiimide to promote migration of the 4-phosphate via a...
We have synthesized phosphatidylinositol 3-phosphate from phosphatidylinositol 4-phosphate by using diisopropylcarbodiimide to promote migration of the 4-phosphate via a cyclic phosphodiester intermediate. The product was isolated by a thin-layer chromatographic method that depends on the ability of phosphatidylinositol 4-phosphate, but not phosphatidylinositol 3-phosphate, to form complexes with boric acid. The final yield of the procedure was 8% phosphatidylinositol 3-phosphate, which was approximately 80% pure. The product was shown to be phosphatidylinositol 3-phosphate by the following criteria: (i) cochromatography with an authentic standard on borate thin-layer chromatography, (ii) cochromatography of the deacylated product with glycerophosphoinositol 3-phosphate on high-performance liquid chromatography, (iii) conversion of the product to phosphatidylinositol by homogeneous phosphatidylinositol 3-phosphate 3-phosphatase, and (iv) deacylation and deglyceration of the product to a compound that comigrates with inositol 1,3-bisphosphate on high-performance liquid chromatography. The availability of mass amounts of phosphatidylinositol 3-phosphate will allow further elaboration of reactions in this recently discovered pathway of phosphatidylinositol metabolism.
Topics: Autoradiography; Chromatography, Thin Layer; Indicators and Reagents; Isomerism; Phosphatidylinositol Phosphates; Phosphatidylinositols; Phosphorus Radioisotopes
PubMed: 1656463
DOI: 10.1073/pnas.88.20.9184 -
The Journal of Physiology Sep 2010Phosphoinositides are a family of minority acidic phospholipids in cell membranes. Their principal role is instructional: they interact with proteins. Each cellular... (Review)
Review
Phosphoinositides are a family of minority acidic phospholipids in cell membranes. Their principal role is instructional: they interact with proteins. Each cellular membrane compartment uses a characteristic species of phosphoinositide. This signature phosphoinositide attracts a specific complement of functionally important, loosely attached peripheral proteins to that membrane. For example, the phosphatidylinositol 4,5-bisphosphate (PIP(2)) of the plasma membrane attracts phospholipase C, protein kinase C, proteins involved in membrane budding and fusion, proteins regulating the actin cytoskeleton, and others. Phosphoinositides also regulate the activity level of the integral membrane proteins. Many ion channels of the plasma membrane need the plasma-membrane-specific PIP(2) to function. Their activity decreases when the abundance of this lipid falls, as for example after activation of phospholipase C. This behaviour is illustrated by the suppression of KCNQ K(+) channel current by activation of M(1) muscarinic receptors; KCNQ channels require PIP(2) for their activity. In summary, phosphoinositides contribute to the selection of peripheral proteins for each membrane and regulate the activity of the integral proteins.
Topics: Animals; Humans; Membrane Lipids; Membrane Proteins; Phosphatidylinositols; Protein Transport
PubMed: 20519312
DOI: 10.1113/jphysiol.2010.192153 -
Nature May 1991Neutrophils activated by the formyl peptide f-Met-Leu-Phe transiently accumulate a small subset of highly polar inositol lipids. A similar family of lipids also appear...
Neutrophils activated by the formyl peptide f-Met-Leu-Phe transiently accumulate a small subset of highly polar inositol lipids. A similar family of lipids also appear in many other cells in response to a range of growth factors and activated oncogenes, and are presumed to be the direct or indirect products of 3-phosphatidylinositol kinase. The structures of these lipids are shown to be phosphatidylinositol 3-phosphate, phosphatidylinositol-(3,4)bisphosphate and phosphatidylinositol-(3,4,5)trisphosphate, and we present evidence that in intact neutrophils a phosphatidyl-inositol-(4,5)bisphosphate-3-kinase seems to be the focal point through which agonists stimulate the formation of 3-phosphorylated inositol lipids.
Topics: Humans; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Kinetics; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Phosphates; Phosphatidylinositol Phosphates; Phosphatidylinositols; Phosphorus Radioisotopes; Radioisotope Dilution Technique
PubMed: 1851250
DOI: 10.1038/351033a0 -
Nature May 1988The responses of mammalian cells to external signals are commonly mediated by intracellular secondary messengers, among which are the breakdown products of...
The responses of mammalian cells to external signals are commonly mediated by intracellular secondary messengers, among which are the breakdown products of phosphatidylinositol 4,5-bisphosphate (PIP2): 1,2-diacylglycerol (DG) and inositol 1,4,5-triphosphate (IP3) (refs 1-7). Although phosphoinositide turnover in the yeast Saccharomyces cerevisiae has been shown to be regulated by glucose and sterol, as yet no definitive function has been ascribed to yeast phosphoinositides. We have recently developed a monoclonal antibody specific for PIP2 and reported that it inhibits mitogenesis of mammalian cells stimulated by platelet-derived growth factor and bombesin. We now report that when introduced into yeast cells by electroporation this antibody inhibits their growth. Furthermore, several yeast mutants with temperature-dependent growth defects are altered in their sensitivity to our antibody and are found to have specific alterations in their phosphoinositide metabolism.
Topics: 1-Phosphatidylinositol 4-Kinase; Genotype; Inositol Phosphates; Kinetics; Mutation; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phosphotransferases; Phosphotransferases (Alcohol Group Acceptor); Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 2835684
DOI: 10.1038/333188a0 -
Biochemical and Biophysical Research... Sep 2006It is well-established that the endoplasmic reticulum is the major site of phosphatidylinositol (PtdIns) synthesis. The PtdIns synthetic ability of other organelles,... (Comparative Study)
Comparative Study
It is well-established that the endoplasmic reticulum is the major site of phosphatidylinositol (PtdIns) synthesis. The PtdIns synthetic ability of other organelles, such as plasma membrane and nucleus, remains controversial. In the present study, we re-examine this question by comparing PtdIns synthesis in isolated cytoplasts (enucleated cells) with that in corresponding karyoplasts (nuclei surrounded by plasma membrane but lacking most cytoplasmic components). We report that cytoplasts are competent to carry out both basal and stimulated PtdIns synthesis as well as polyphosphoinositide hydrolysis, while karyoplasts can neither synthesize PtdIns nor hydrolyze phosphoinositides in response to agonists. The karyoplasts are, however, capable of synthesizing phosphatidylcholine (PtdCho), as previously reported. From these data, we conclude that PtdIns synthesis is limited to cytoplasmic components, and cannot be sustained by either plasma membrane or nucleus under conditions that permit robust PtdCho synthesis.
Topics: Animals; Cell Line, Tumor; Mammary Neoplasms, Animal; Phosphatidylinositols; Rats; Subcellular Fractions
PubMed: 16904631
DOI: 10.1016/j.bbrc.2006.07.196 -
Chemistry and Physics of Lipids Jan 2005The aim of the present study was to characterize a new lipid detected in the opportunistic pathogen Corynebacterium amycolatum. It was identified as...
The aim of the present study was to characterize a new lipid detected in the opportunistic pathogen Corynebacterium amycolatum. It was identified as acyl-phosphatidylinositol (acyl-PI), and revealed as a mixture of homologues compounds by electrospray ionization mass spectrometry, with pseudomolecular ions, (M-H)-, observed at 1099 (the major one) 1113, and 1127. Acyl-PI exclusively contained octadecenoyl on the inositol moiety (as 3-O-acyl), an unsaturated fatty acyl (mostly octadecenoyl) at sn-1 position of the glycerol and a saturated fatty acyl (mainly hexadecanoyl) at the sn-2 position. Acyl-PI constitutes a new natural substance and seems to be unique among the phospholipids of C. amycolatum. Other more complex molecules, previously undetected, and assigned in this work to several acyl forms of phosphatidylinositol trimannosides, lacked octadecenoyl in their polar heads. The present study reveals the existence of acyl-PI in C. amycolatum as rather unexpected finding and, additionally, gives evidence for the ability of this species to synthesize a great variety of inositol-containing phospholipids.
Topics: Corynebacterium; Molecular Structure; Phosphatidylinositols; Spectrometry, Mass, Electrospray Ionization
PubMed: 15589223
DOI: 10.1016/j.chemphyslip.2004.08.001 -
Journal of Cell Science Dec 2020Phosphoinositides (PIPs) are a dynamic family of lipids that execute diverse roles in cell biology. PIP levels are regulated by numerous enzymes, but our understanding...
Phosphoinositides (PIPs) are a dynamic family of lipids that execute diverse roles in cell biology. PIP levels are regulated by numerous enzymes, but our understanding of how these enzymes are controlled in space and time is incomplete. One role of the PIP phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P] is to anchor the cytokinetic ring (CR) to the plasma membrane (PM) in While examining potential PI(4,5)P-binding proteins for roles in CR anchoring, we identified the dual pleckstrin homology (PH) domain-containing protein Opy1. Although related proteins are implicated in PIP regulation, we found no role for Opy1 in CR anchoring, which would be expected if it modulated PM PI(4,5)P levels. Our data indicate that although Opy1 senses PM PI(4,5)P levels and binds to the phosphatidylinositol 4-phosphate 5-kinase (PI5-kinase) Its3, Opy1 does not regulate Its3 kinase activity or PM PI(4,5)P levels, a striking difference from its homolog. However, overexpression of Opy1 resulted in cytokinesis defects, as might be expected if it sequestered PI(4,5)P Our results highlight the evolutionary divergence of dual PH domain-containing proteins and the need for caution when interpreting results based on their overexpression.This article has an associated First Person interview with the first author of the paper.
Topics: Cell Membrane; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositol Phosphates; Phosphatidylinositols; Schizosaccharomyces
PubMed: 33172987
DOI: 10.1242/jcs.247973 -
Developmental Cell Nov 2011Polyphosphoinositides are lipid signaling molecules generated from phosphatidylinositol (PtdIns) with critical roles in vesicular trafficking and signaling. It is poorly...
Polyphosphoinositides are lipid signaling molecules generated from phosphatidylinositol (PtdIns) with critical roles in vesicular trafficking and signaling. It is poorly understood where PtdIns is located within cells and how it moves around between membranes. Here we identify a hitherto-unrecognized highly mobile membrane compartment as the site of PtdIns synthesis and a likely source of PtdIns of all membranes. We show that the PtdIns-synthesizing enzyme PIS associates with a rapidly moving compartment of ER origin that makes ample contacts with other membranes. In contrast, CDP-diacylglycerol synthases that provide PIS with its substrate reside in the tubular ER. Expression of a PtdIns-specific bacterial PLC generates diacylglycerol also in rapidly moving cytoplasmic objects. We propose a model in which PtdIns is synthesized in a highly mobile lipid distribution platform and is delivered to other membranes during multiple contacts by yet-to-be-defined lipid transfer mechanisms.
Topics: Animals; COS Cells; Cell Membrane; Chlorocebus aethiops; Diacylglycerol Cholinephosphotransferase; Endoplasmic Reticulum; HEK293 Cells; Humans; Organelles; Phosphatidylinositols; Signal Transduction
PubMed: 22075145
DOI: 10.1016/j.devcel.2011.09.005 -
The Biochemical Journal Apr 1963
Topics: Animals; Brain; Inositol; Phosphatidylinositol Phosphates; Phosphatidylinositols; Rats
PubMed: 13981209
DOI: 10.1042/bj0870136