Enzymatic fluorometric assay for myo-inositol trisphosphate.
The determination of myo-inositol trisphosphate by an enzymatic fluorometric assay is presented. The method involves the acid extraction of water-soluble inositol polyphosphates followed by separation by anion-exchange chromatography. Samples are subsequently neutralized by passage over a Dowex Cl- resin and elution with lithium chloride. Samples are then desalted with ethanol. Following dephosphorylation with alkaline phosphatase, free myo-inositol is measured enzymatically via the NAD-dependent oxidation to scyllo-inosose with myo-inositol dehydrogenase. The efficiency of recovery, assay specificity, and an application to the measurement of inositol polyphosphates in hormone-stimulated tissue are discussed.
D000469:Alkaline Phosphatase; D000818:Animals; D002852:Chromatography, Ion Exchange; D015544:Inositol 1,4,5-Trisphosphate; D007295:Inositol Phosphates / Q000032:analysis*; D007668:Kidney / Q000032:analysis; D008291:Malate Dehydrogenase; D010084:Oxidation-Reduction; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013401:Sugar Alcohol Dehydrogenases; D013403:Sugar Phosphates / Q000032:analysis*
D007295:Inositol Phosphates; D013403:Sugar Phosphates; D015544:Inositol 1,4,5-Trisphosphate; D013401:Sugar Alcohol Dehydrogenases; C021448:myo-inositol 2-dehydrogenase; D008291:Malate Dehydrogenase; D000469:Alkaline Phosphatase