Abstract
Phosphatidylinositol (PtdIns) synthase is a lipid-synthesizing enzyme responsible for the synthesis of the phospholipid, PtdIns. Its enzymatic properties have been studied in in vitro assays using either membrane-enriched fractions or the purified protein in reconstituted lipid vesicles as a source of enzyme. More recently the specificities in terms of substrate preferences have also been studied using the recombinant protein expressed in Escherichia coli. This chapter deals with the purification of membranes as a source of PtdIns synthase before focusing on the in vitro assays of the enzymatic activities of the protein and, briefly, on the analysis of the product.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Sumida S, Mudd JB (1970) The structure and biosynthesis of phosphatidylinositol in cauliflower inflorescence. Plant Physiol 45:712–718
Moore TS, Lord JM, Kagawa T et al (1973) Enzymes of phospholipid metabolism in endoplasmic reticulum of castor bean endosperm. Plant Physiol 52:50–53
Sexton JC, Moore TS (1978) Phosphatidylinositol synthesis in castor bean endosperm. Plant Physiol 62:978–980
Collin S, Justin A-M, Cantrel C et al (1999) Identification of AtPIS, a phosphatidylinositol synthase from Arabidopsis. Eur J Biochem 262:652–658
Blouin A, Lavezzi T, Moore TS (2003) Membrane lipid biosynthesis in Chlamydomona sreinhardtii. Partial characterization of CDP-diacylglycerol: myo-inositol 3-phosphatidyltransferase. Plant Physiol Biochem 41:11–16
Justin A-M, Kader J-C, Collin S (2003) Synthetic capacity of Arabidopsis phosphatidylinositol synthase 1 expressed in E. coli. Biochim Biophys Acta 1634:52–60
Davy de Virville J, Brown S, Cochet F et al (2010) Assessment of mitochondria as a compartment for phosphatidylinositol synthesis in Solanum tuberosum. Plant Physiol Biochem 48:952–960
Justin A-M, Hmyene A, Kader J-C et al (1994) Compared selectivities of the phosphatidylinositol synthase from maize coleoptiles either in microsomal membranes or after solubilization. Biochim Biophys Acta 1255:161–166
Sexton JC, Moore TS (1981) Phosphatidylinositol synthesis by a Mn-dependent exchange enzyme in castor bean endosperm. Plant Physiol 68:18–22
Löfke C, Ischebeck T, König S et al (2008) Alternative metabolic fates of phosphatidylinositol produced by phosphatidylinositol synthase isoforms in Arabidopsis thaliana. Biochem J 413:115–124
Ohta A, Obara T, Asami Y et al (1985) Molecular cloning of the cls gene responsible for cardiolipin synthesis in E. coli and phenotypic consequences of its amplification. J Bacteriol 163:506–514
Lowry OH, Rosebrough NJ, Farr AL et al (1952) Protein measurements with the Folin reagent. J Biol Chem 193:265–275
Vaultier MN, Cantrel C, Vergnolle C et al (2006) Desaturase mutants reveal that membrane rigidification acts as a cold perception mechanism upstream of the diacylglycerol kinase pathway in Arabidopsis cells. FEBS Lett 580:418–4223
Ahmed HA (1993) Analysis of Phospholipids by high performance liquid chromatography. In: Graham J, Higgins J (eds) Biomembrane Protocols: I. Isolation and Analysis, Methods in Molecular Biology, Springer Protocols, vol. 19, pp. 159–177
Quinn PJ, Rainteau D, Wolf C (2009) Lipidomics of the Red Cell in Diagnosis of Human Disorders. In: Amstrong D (ed) Lipidomics: vol. 1: Methods and Protocols, Methods in MolecularBiology, Springer Protocols, vol. 579, pp. 127–159
Vincent P, Maneta-Peyret L, Cassagne C et al (2001) Phosphatidylserine delivery to endoplasmic reticulum-derived vesicles of plant cells depends on two biosynthetic pathways. FEBS Lett 498:32–36
Justin A-M, Kader J-C, Collin S (2002) Phosphatidylinositol synthesis and exchange of the inositol head are catalysed by the single phosphatidylinositol synthase 1 from Arabidopsis. Eur J Biochem 269:2347–2352
Acknowledgments
The authors wish to thank E. Ruelland for his careful reading of the manuscript. Financial support from the Université Pierre et Marie Curie via the French Ministère de l’Enseignement Supérieur et de la Recherche, as well as from the C.N.R.S., is gratefully acknowledged.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Collin, S., Cochet, F. (2013). Phosphatidylinositol Synthase Activity from Plant Membrane Fractions and from E. coli- Expressed Recombinant Peptides. In: Munnik, T., Heilmann, I. (eds) Plant Lipid Signaling Protocols. Methods in Molecular Biology, vol 1009. Humana, Totowa, NJ. https://doi.org/10.1007/978-1-62703-401-2_14
Download citation
DOI: https://doi.org/10.1007/978-1-62703-401-2_14
Published:
Publisher Name: Humana, Totowa, NJ
Print ISBN: 978-1-62703-400-5
Online ISBN: 978-1-62703-401-2
eBook Packages: Springer Protocols