Abstract
Data on the production of inositol phosphates is a useful complement to measurements of intracellular Ca2+. The basic principle is labeling of the inositol lipids by growing the appropriate cell line in culture in the presence of [3H]inositol for 2–3 days to reach labeling equilibrium. Lithium ions at 10 mM inhibits the degradation of inositol phosphates to free inositol and is used to trap the inositol in the inositol polyphosphate forms. Inositol phosphates can be separated with ease from free inositol by using anion exchange chromatography. A method capable of easily processing approximately 40–60 samples in a single day is presented.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Rhee SG (2001) Regulation of phosphoinositide-specific phospholipase C. Annu Rev Biochem 70:281–312
Rebecchi MJ, Pentyala SN (2000) Structure, function, and control of phosphoinositide-specific phospholipase C. Physiol Revs 80:1291–1335
Thomas GMH, Cunningham E, Fensome A, Ball A, Totty NF, Troung O, Hsuan JJ, Cockcroft S (1993) An essential role for phosphatidylinositol transfer protein in phospholipase C-mediated inositol lipid signalling. Cell 74:919–928
Kauffmann-Zeh A, Thomas GMH, Ball A, Prosser S, Cunningham E, Cockcroft S, Hsuan JJ (1995) Requirement for phosphatidylinositol transfer protein in epidermal growth factor signalling. Science 268:1188–1190
Allen V, Swigart P, Cheung R, Cockcroft S, Katan M (1997) Regulation of inositol lipid-specific phospholipase Cδ by changes in Ca2+ ion concentrations. Biochem J 327:545–552
Cunningham E, Tan SW, Swigart P, Hsuan J, Bankaitis V, Cockcroft S (1996) The yeast and mammalian isoforms of phosphatidylinositol transfer protein can all restore phospholipase C-mediated inositol lipid signalling in cytosol-depleted RBL-2H3 and HL60 cells. Proc Natl Acad Sci U S A 93:6589–6593
Skippen A, Jones DH, Morgan CP, Li M, Cockcroft S (2002) Mechanism of ADP ribosylation-factor-stimulated phosphatidylinositol 4,5-bisphosphate synthesis in HL60 cells. J Biol Chem. 277:5823–5831
Tatham PER, Gomperts BD (1990) Cell permeabilisation. In: Siddle K, Hutton JC (eds) Peptide hormones—a practical approach. IRL, Oxford, England, pp 257–269
Bird IM (1998) Phosphoinositidase C activation assay III. Methods in molecular biology 105: phospholipid signaling protocols. Bird IM (ed) Humana Press Inc, Totowa, NJ, pp 25–45.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Skippen, A., Swigart, P., Cockcroft, S. (2013). Measurement of Phospholipase C by Monitoring Inositol Phosphates Using [3H]Inositol Labeling Protocols in Permeabilized Cells. In: Lambert, D., Rainbow, R. (eds) Calcium Signaling Protocols. Methods in Molecular Biology, vol 937. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-086-1_9
Download citation
DOI: https://doi.org/10.1007/978-1-62703-086-1_9
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-085-4
Online ISBN: 978-1-62703-086-1
eBook Packages: Springer Protocols