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Simple synthesis of (32)P-labelled inositol hexakisphosphates for study of phosphate transformations
BACKGROUND AND AIMS: In 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 e...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Springer International Publishing
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5984642/ https://www.ncbi.nlm.nih.gov/pubmed/29880988 http://dx.doi.org/10.1007/s11104-017-3315-9 |
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author | Whitfield, Hayley Riley, Andrew M. Diogenous, Soulla Godage, Himali Y. Potter, Barry V. L. Brearley, Charles A. |
author_facet | Whitfield, Hayley Riley, Andrew M. Diogenous, Soulla Godage, Himali Y. Potter, Barry V. L. Brearley, Charles A. |
author_sort | Whitfield, Hayley |
collection | PubMed |
description | BACKGROUND AND AIMS: In 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 (32)P would greatly enable study of transformation of soil inositol phosphates when combined with robust HPLC separations of different inositol phosphates. METHODS: We employed the enzyme inositol pentakisphosphate 2-kinase, IP5 2-K, to transfer phosphate from [γ-(32)P]ATP to axial hydroxyl(s) of myo-, neo- and 1D-chiro-inositol phosphate substrates. RESULTS: (32)P-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. CONCLUSIONS: We developed enzymic approaches that allow the synthesis of labeled myo-inositol 1,[(32)P]2,3,4,5,6-hexakisphosphate; neo-inositol 1,[(32)P]2,3,4,[(32)P]5,6–hexakisphosphate and 1D-chiro-inositol [(32)P]1,2,3,4,5,[(32)P]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 MATERIAL: The online version of this article (doi:10.1007/s11104-017-3315-9) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-5984642 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Springer International Publishing |
record_format | MEDLINE/PubMed |
spelling | pubmed-59846422018-06-28 Simple synthesis of (32)P-labelled inositol hexakisphosphates for study of phosphate transformations Whitfield, Hayley Riley, Andrew M. Diogenous, Soulla Godage, Himali Y. Potter, Barry V. L. Brearley, Charles A. Plant Soil Regular Article BACKGROUND AND AIMS: In 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 (32)P would greatly enable study of transformation of soil inositol phosphates when combined with robust HPLC separations of different inositol phosphates. METHODS: We employed the enzyme inositol pentakisphosphate 2-kinase, IP5 2-K, to transfer phosphate from [γ-(32)P]ATP to axial hydroxyl(s) of myo-, neo- and 1D-chiro-inositol phosphate substrates. RESULTS: (32)P-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. CONCLUSIONS: We developed enzymic approaches that allow the synthesis of labeled myo-inositol 1,[(32)P]2,3,4,5,6-hexakisphosphate; neo-inositol 1,[(32)P]2,3,4,[(32)P]5,6–hexakisphosphate and 1D-chiro-inositol [(32)P]1,2,3,4,5,[(32)P]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 MATERIAL: The online version of this article (doi:10.1007/s11104-017-3315-9) contains supplementary material, which is available to authorized users. Springer International Publishing 2017-06-27 2018 /pmc/articles/PMC5984642/ /pubmed/29880988 http://dx.doi.org/10.1007/s11104-017-3315-9 Text en © The Author(s) 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. |
spellingShingle | Regular Article Whitfield, Hayley Riley, Andrew M. Diogenous, Soulla Godage, Himali Y. Potter, Barry V. L. Brearley, Charles A. Simple synthesis of (32)P-labelled inositol hexakisphosphates for study of phosphate transformations |
title | Simple synthesis of (32)P-labelled inositol hexakisphosphates for study of phosphate transformations |
title_full | Simple synthesis of (32)P-labelled inositol hexakisphosphates for study of phosphate transformations |
title_fullStr | Simple synthesis of (32)P-labelled inositol hexakisphosphates for study of phosphate transformations |
title_full_unstemmed | Simple synthesis of (32)P-labelled inositol hexakisphosphates for study of phosphate transformations |
title_short | Simple synthesis of (32)P-labelled inositol hexakisphosphates for study of phosphate transformations |
title_sort | simple synthesis of (32)p-labelled inositol hexakisphosphates for study of phosphate transformations |
topic | Regular Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5984642/ https://www.ncbi.nlm.nih.gov/pubmed/29880988 http://dx.doi.org/10.1007/s11104-017-3315-9 |
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