6 research outputs found
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HPLC Separation of Inositol Polyphosphates
High performance liquid chromatography (H PLC) is an essential analytical tool in the study of the large number of inositol phosphate isomers. This chapter focuses on the separation of inositol polyphosphates from [H-3]myo-inositol labeled tissues and cells.
We review the different HPLC columns that have been used to separate inositol phosphates and their advantages and disadvantages. We describe important elements of sample preparation for effective separations and give examples of how changing factors, such as pH, can considerably improve the resolving ability of the HPLC chromatogram
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Diphosphosinositol Polyphosphates and Energy Metabolism: Assay for ATP/ADP Ratio
Several inositol compounds undergo rapid cycles of phosphorylation and dephosphorylation. These cycles at.: dependent on ATP and energy metabolism. Therefore, interfering with the cellular energy metabolism can change the concentration of rapidly turning over inositols. Many pharmacological inhibitors, apart from their intended action, also affect the energy metabolism of the cells and lower ATP. This can unspecifically influence rapidly turning over inositol phosphates. Thus, the ATP concentration should be checked when reduced inositol phosphates are observed after application of pharmacological inhibitors.
A luminescence-based assay for the measurement of ATP and ADP is described. ATP is measured luminometrically using firefly luciferase. Detection of ADP is performed in a two-step enzymatic procedure: (1) The sample ATP is degraded to AMP and (2) ADP is phosphorylated to ATP, which can then be measured luminometrically. This method gives a better signal-to-noise ratio than other methods that do not degrade the sample ATP, but convert ADP directly to ATP and then measure the sum of ATP plus ADP