Simple synthesis of 32P-labelled inositol hexakisphosphates for study of phosphate transformations

Abstract

Background and aimsIn many soils inositol hexakisphosphate in its various forms is as abundant as inorganic phosphate. The organismal and geochemical processes that exchange phosphate between inositol hexakisphosphate and other pools of soil phosphate are poorly defined, as are the organisms and enzymes involved. We rationalized that simple enzymic synthesis of inositol hexakisphosphate labeled with 32P would greatly enable study of transformation of soil inositol phosphates when combined with robust HPLC separations of different inositol phosphates.MethodsWe employed the enzyme inositol pentakisphosphate 2-kinase, IP5 2-K, to transfer phosphate from [γ-32P]ATP to axial hydroxyl(s) of myo-, neo- and 1D-chiro-inositol phosphate substrates.Results 32P-labeled inositol phosphates were separated by anion exchange HPLC with phosphate eluents. Additional HPLC methods were developed to allow facile separation of myo-, neo-, 1D-chiro- and scyllo-inositol hexakisphosphate on acid gradients.ConclusionsWe developed enzymic approaches that allow the synthesis of labeled myo-inositol 1,[32P]2,3,4,5,6-hexakisphosphate; neo-inositol 1,[32P]2,3,4,[32P]5,6–hexakisphosphate and 1D-chiro-inositol [32P]1,2,3,4,5,[32P]6-hexakisphosphate. Additionally, we describe HPLC separations of all inositol hexakisphosphates yet identified in soils, using a collection of soil inositol phosphates described in the seminal historic studies of Cosgrove, Tate and coworkers. Our study will enable others to perform radiotracer experiments to analyze fluxes of phosphate to/from inositol hexakisphosphates in different soils.Electronic supplementary materialThe online version of this article (doi:10.1007/s11104-017-3315-9) contains supplementary material, which is available to authorized users.

An ATP-responsive metabolic cassette comprised of inositol tris/tetrakisphosphate kinase 1 (ITPK1) and inositol pentakisphosphate 2-kinase (IPK1) buffers diphosphosphoinositol phosphate levels

“…We employed anion exchange chromatography on an acid- and hydroxide-stable quaternary amine-functionalized latex (CarboPac PA200, Dionex) as we have done before [ 27 ]. This chemistry allows the use of the widest range of eluents.…”

“…HPLC was performed according to Whitfield et al [27] using either 0.6 M methanesulfonic acid or 0.8 M HCl in buffer reservoirs. Inositol phosphates were detected as ferric complexes by UV detection at 290 nm [28].…”

A Fluorescent Probe Identifies Active Site Ligands of Inositol Pentakisphosphate 2-Kinase

“…the kinase activity of AtIP5 2-K , while D-chiro-inositol 1,3,4,6-tetrakisphosphate is not 32 . The first possesses axial hydroxyls on the 2-and 5-positions, the second has axial hydroxyls on the 1-and 6-positions, while the third has no axial hydroxyls 31 .…”

“…K in vivo, we radiolabeled Arabidopsis thaliana seedlings with32 P orthophosphate in media containing purpurogallin, 5,6,7,8,4'-pentahydroxy flavone or chaetochromin and quantified inositol phosphates and ATP by HPLC. In labeled Arabidopsis, ATP and InsP 6 are by far the strongest labeled peaks, other than unincorporated inorganic phosphate, while myo-Ins(1,3,4,5,6)P 5 is the least strongly labeled of InsP 5 s 38 .…”

Organic phosphorus: potential solutions for phosphorus security

“…The accepted manuscripts cover an appropriate range of topics related to organic phosphorus. This issue contains papers that we can class as 'reviews' (two papers, George et al 2017 andMenezesBlackburn et al 2017), 'plant centric' (Li et al 2017 andDarch et al 2017), 'soil centric' (three papers, Stahr et al 2017;Missong et al 2017 andSpain et al 2018) 'microbial centric' (two papers Nassal et al 2017 andGaiero et al 2017), 'analytical' (two papers, Adams et al 2017 andWhitfield et al 2017) and 'systems' (two papers Neal et al 2017). An additional historical overview paper has also been published from the Workshop (Haygarth et al 2018).…”