Abstract
A nonradioactive high-performance anion-exchange chromatographic method based on MDD-HPLC (Mayr Biochem. J. 254:585–591, 1988) was developed for the separation of inositol hexakisphosphate (InsP 6, phytic acid) and most isomers of pyrophosphorylated inositol phosphates, such as diphosphoinositol pentakisphosphate (PPInsP 5 or InsP 7) and bis-diphosphoinositol tetrakisphosphate (bisPPInsP 4 or InsP 8). With an acidic elution, the anion-exchange separation led to the resolution of four separable PPInsP 5 isomers (including pairs of enantiomers) into three peaks and of nine separable bisPPInsP 4 isomers into nine peaks. The whole separation procedure was completed within 20–36 min after optimization. Reference standards of all bisPPInsP 4 isomers were generated by a nonenzymatic shotgun synthesis from InsP 6. Hereby, the phosphorylation was brought about nonenzymatically when concentrated InsP 6 bound to the solid surface of anion-exchange beads was incubated with creatine phosphate under optimal pH conditions. From the mixture of pyrophosphorylated InsP 6 derivatives containing all theoretically possible isomers of PPInsP 5, bisPPInsP 4, and also some isomers of trisPPInsP 3, isomers were separated by anion-exchange chromatography and fractions served as reference standards of bisPPInsP 4 isomers for further investigation. Their isomeric nature could be partly assigned by comparison with position specifically synthesized or NMR-characterized purified protozoan reference compounds and partly by limited hydrolysis to PPInsP 5 isomers. By applying this nonradioactive analysis technique to cellular studies, the isomeric nature of the major bisPPInsP 4 in mammalian cells could be identified without the need to obtain sufficient material for NMR analysis.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Mayr, G.W. (1988) A novel metal-dye detection system permits picomolar-range h.p.l.c. analysis of inositol polyphosphates from non-radioactively labelled cell or tissue specimens. Biochem. J. 254, 585–591.
Pittet, D., Schlegel, W., Lew, D., Monod, A., and Mayr, G. (1989) Mass changes in inositol tetrakis- and pentakisphosphate isomers induced by chemotactic peptide stimulation in HL-60 cells. J. Biol. Chem. 264, 18489–18493.
Lorke, D.E., Gustke, H., and Mayr, G.W. (2004) An optimized fixation and extraction technique for high resolution of inositol phosphate signals in rodent brain. Neurochem. Res. 29, 1887–1896.
Martin, J.B., Foray, M.F., Klein, G., and Satre, M. (1987) Identification of inositol hexaphosphate in 31P-NMR spectra of Dictyostelium discoideum amoebae. Relevance to intracellular pH determination. Biochim. Biophys. Acta. 931, 16–25.
Safrany, S.T., Caffrey, J.J., Yang, X., and Shears, S.B. (1999) Diphosphoinositol polyphosphates: the final frontier for inositide research? Biol. Chem. 380, 945–951.
Menniti, F., Miller, R., Putney, J., Jr, and Shears, S. (1993) Turnover of inositol polyphosphate pyrophosphates in pancreatoma cells. J. Biol. Chem. 268, 3850–3856.
Europe-Finner, G.N., Gammon, B., Wood, C.A., and Newell, P.C. (1989) Inositol tris- and polyphosphate formation during chemotaxis of Dictyostelium. J. Cell Sci. 93, 585–592.
Mayr, G.W., Radenberg, T., Thiel, U., Vogel, G., and Stephens, L.R. (1992) Phosphoinositol disphosphates: non-enzymic formation in vitro and occurence in vivo in the cellular slime mold Dictyostelium. Carbohydr. Res. 234, 247–262.
Stephens, L., Radenberg, T., Thiel, U., Vogel, G., Khoo, K.H., Dell, A., Jackson, T.R., Hawkins, P.T., Mayr, G.W., Stephens, L.R., Stanley, A.F., Moore, T., Poyner, D.R., Morris, P.J., Hanley, M.R., Kay, R.R., Irvine, R.F., Laussmann, T., Eujen, R., Weisshuhn, C.M., Martin, J.B., Bakker-Grunwald, T., Klein, G., Reddy, K.M., Reddy, K.K., and Falck, J.R. (1993) The detection, purification, structural characterization, and metabolism of diphosphoinositol pentakisphosphate(s) and bisdiphosphoinositol tetrakisphosphate(s) myo-inositol pentakisphosphates. J. Biol. Chem. 268, 4009–4015.
Albert, C., Safrany, S.T., Bembenek, M.E., Reddy, K.M., Reddy, K., Falck, J., Brocker, M., Shears, S.B., and Mayr, G.W. (1997) Biological variability in the structures of diphosphoinositol polyphosphates in Dictyostelium discoideum and mammalian cells. Biochem. J. 327, 553–560.
Laussmann, T., Reddy, K.M., Reddy, K.K., Falck, J.R., and Vogel, G. (1997) Diphospho-myo-inositol phosphates from Dictyostelium identified as D-6-diphospho-myo-inositol pentakisphosphate and D-5,6-bisdiphospho-myo-inositol tetrakisphosphate. Biochem. J. 322, 31–33.
Laussmann, T., Hansen, A., Reddy, K.M., Reddy, K.K., Falck, J.R., and Vogel, G. (1998) Diphospho-myo-inositol phosphates in Dictyostelium and Polysphondylium: identification of a new bisdiphospho-myo-inositol tetrakisphosphate. FEBS Lett. 426, 145–150.
Martin, J.-B., Bakker-Grunwald, T., and Klein, G. (1993) 31P-NMR analysis of Entamoeba histolytica. Occurrence of high amounts of two inositol phosphates. Eur. J. Biochem. 214, 711–718.
Bennett, M., Onnebo, S.M., Azevedo, C., and Saiardi, A. (2006) Inositol pyrophosphates: metabolism and signaling. Cell Mol. Life Sci. 63, 552–564.
York, J.D. (2006) Regulation of nuclear processes by inositol polyphosphates. Biochim. Biophys. Acta. 1761, 552–559.
Irvine, R.F. (2006) Nuclear inositide signalling – expansion, structures and clarification. Biochim. Biophys. Acta. 1761, 505–508.
Luo, H.R., Saiardi, A., Yu, H., Nagata, E., Ye, K., and Snyder, S.H. (2002) Inositol pyrophosphates are required for DNA hyperrecombination in protein kinase C1 mutant yeast. Biochemistry. 41, 2509–2515.
Luo, H.R., Huang, Y.E., Chen, J.C., Saiardi, A., Iijima, M., Ye, K., Huang, Y., Nagata, E., Devreotes, P., and Snyder, S.H. (2003) Inositol pyrophosphates mediate chemotaxis in Dictyostelium via pleckstrin homology domain-PtdIns(3,4,5)P3 interactions. Cell. 114, 559–572.
York, S.J., Armbruster, B.N., Greenwell, P., Petes, T.D., and York, J.D. (2005) Inositol diphosphate signaling regulates telomere length. J. Biol. Chem. 280, 4264–4269.
Saiardi, A., Resnick, A.C., Snowman, A.M., Wendland, B., Snyder, S.H., Bhandari, R., Pesesse, X., Choi, K., Zhang, T., Shears, S.B., Luo, H.R., Huang, Y.E., Chen, J.C., Iijima, M., Ye, K., Huang, Y., Nagata, E., Devreotes, P., El Alami, M., Messenguy, F., Scherens, B., Dubois, E., Sciambi, C., McCaffery, J.M., Yu, H., Menniti, F.S., Miller, R.N., and Putney, J.W., Jr. (2005) Inositol pyrophosphates regulate cell death and telomere length through phosphoinositide 3-kinase-related protein kinases phosphorylation of proteins by inositol pyrophosphates. Proc. Natl. Acad. Sci. USA 102, 1911–1914.
York, J.D., Odom, A.R., Murphy, R., Ives, E.B., and Wente, S.R. (1999) A phospholipase C-dependent inositol polyphosphate kinase pathway required for efficient messenger RNA export. Science. 285, 96–100.
Illies C, Gromada J, Fiume R, Leibiger B, Yu J, Juhl K, Yang SN, Barma DK, Falck JR, Saiardi A, Barker CJ, Berggren PO. (2007) Requirement of inositol pyrophosphates for full exocytotic capacity in pancreatic beta cells. Science. 318, 1299–1302.
Saiardi, A., Bhandari, R., Resnick, A.C., Snowman, A.M., and Snyder, S.H. (2004) Phosphorylation of proteins by inositol pyrophosphates. Science. 306, 2101–2105.
Choi, K., Mollapour, E., and Shears, S.B. (2005) Signal transduction during environmental stress: InsP(8) operates within highly restricted contexts. Cell. Signal. 17, 1533–1541.
Pesesse, X., Choi, K., Zhang, T., and Shears, S.B. (2004) Signaling by higher inositolpolyphosphates: hyperosmotic stress acutely and selectively activates synthesis of bis-diphosphoinositol tetrakisphosphate (“InsP8”). J. Biol. Chem. 279, 43378–43381.
Draskovic, P., Saiardi, A., Bhandari, R., Burton, A., Ilc, G., Kovacevic, M., Snyder, S.H., and Podobnik, M. (2008) Inositol hexakisphosphate kinase products contain diphosphate and triphosphate groups. Chem. Biol. 15, 274–286.
Segel, I.H. (1976) Biochemical calculations: how to solve mathematical problems in general biochemistry. 2nd ed. New York: John Wiley & Sons Inc., pp. 15.
Lide, D.R. (2006) CRC Handbook Chemistry and Physics. 87th ed: Taylor & Francis Group, New York, pp. 8–41.
Lanzetta, P.A., Alvarez, L.J., Reinach, P.S., and Candia, O.A. (1979) An improved assay for nanomole amounts of inorganic phosphate. Anal. Biochem. 100, 95–97.
Acknowledgments
The authors would like to thank Professor Gunter Vogel (Wuppertal, Germany) for providing the 1/3,5-bisPPInsP 4 isomer from Polysphondylium, and Professor J.R. Falck (UT Southwestern, Dallas, USA) for providing a synthetic 2,5-bisPPInsP 4 sample and pure PPInsP 5 isomers. Parts of the MDD-HPLC analysis have been performed by Bettina Serreck, whose technical support is highly acknowledged.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Lin, H., Lindner, K., Mayr, G.W. (2010). Synthesis and Nonradioactive Micro-analysis of Diphosphoinositol Phosphates by HPLC with Postcolumn Complexometry. In: Barker, C. (eds) Inositol Phosphates and Lipids. Methods in Molecular Biology, vol 645. Humana Press. https://doi.org/10.1007/978-1-60327-175-2_7
Download citation
DOI: https://doi.org/10.1007/978-1-60327-175-2_7
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60327-174-5
Online ISBN: 978-1-60327-175-2
eBook Packages: Springer Protocols