A femtomole-sensitivity mass assay for inositol hexakisphosphate.
Inositol hexakisphosphate (InsP(6)) is an important component of cells, and its mass levels are usually assayed by either (a) equilibrium labelling of cell cultures with radiolabelled inositol or (b) by a variety of mass assays of differing sensitivities and ambiguities. Here, we describe a mass assay for InsP(6) that is based on phosphorylating InsP(6) with [(32)P]-ATP to 5-(PP)InsP(5) using a recombinant Giardia InsP(6) kinase and quantification of the radiolabelled 5-[(32)P](PP)InsP(5) product by anion exchange HPLC with an internal [(3)H]-(PP)InsP(5) standard. Interference with the enzyme reaction by other factors in the tissue extract is corrected for by assay of identical aliquots of tissue spiked with known amounts of InsP(6). This assay only measures InsP(6) (and not other inositol phosphates), and although it is simple in principle and requires no dedicated or specialised equipment, it is quite time-consuming. But the assay is unambiguous and is capable of quantifying accurately as little as 10 fmol of InsP(6) in a cell extract.
Letcher
Andrew J
AJ
Department of Pharmacology, University of Cambridge, Cambridge, UK.
Schell
Michael J
MJ
Irvine
Robin F
RF
D000255:Adenosine Triphosphate / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D057075:Enzyme Assays / Q000191:economics / Q000379:methods*; D005872:Giardia / Q000201:enzymology*; D010761:Phosphorus Radioisotopes / Q000378:metabolism; D010766:Phosphorylation; D010833:Phytic Acid / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012680:Sensitivity and Specificity
D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins; D010833:Phytic Acid; D000255:Adenosine Triphosphate