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.Results32P-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.
Highlights
We 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-chiroinositol [32P]1,2,3,4,5,[32P]6-hexakisphosphate
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
It is clear from the foregoing that studies of soil phosphate transformations, those arising from input of myo-inositol hexakisphosphate from plant sources, would be greatly enabled by the provision of 32P or 33P-labelled myo-inositol hexakisphosphate, and, of other inositol hexakisphosphates
Summary
In consideration of the different forms of inositol hexakisphosphate identified in soils: 1D-chiro-, myo-, neo- and scyllo- ((Anderson 1964; Anderson and Malcolm 1974; Baker 1974, cited in Turner et al 2002; Cosgrove 1962, 1963, 1966, 1969a, b; Cosgrove and Tate 1963; Halstead and Anderson 1970; L’Annunziata 1975; L’Annunziata and Fuller 1971; L’Annunziata et al 1972; reviewed, Cosgrove 1980); Irving and Cosgrove 1982), it remains unclear what the biotic or abiotic origins of D-chiro-, neoand scyllo-inositol phosphates are (L’Annunziata 2007; Turner and Richardson 2004; Turner et al 2002). With labelled inositol hexakisphosphates and an increasing literature on the ‘pathways’ of myo-inositol hexakisphosphate degradation by phytases of different classes; cysteine phytase, histidine acid phytase, purple-acid phytase, βpropeller phytase (Konietzny and Greiner 2002), it would be possible to begin to describe ‘pathways’ of inositol hexakisphosphate turnover in soils and the contribution of different organisms to that turnover. With these thoughts in mind, we have sought to synthesize 32P-labeled inositol hexakisphosphates by enzymic means.
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