Letcher Andrew J, Schell Michael J, Irvine Robin F
Department of Pharmacology, University of Cambridge, Cambridge, UK.
Methods Mol Biol. 2010;645:61-71. doi: 10.1007/978-1-60327-175-2_4.
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)PInsP(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.
肌醇六磷酸（InsP(6)）是细胞的重要组成部分，其含量水平通常通过以下两种方法测定：（a）用放射性标记的肌醇对细胞培养物进行平衡标记；（b）通过多种灵敏度和不确定性各异的质量分析法。在此，我们描述了一种针对InsP(6)的质量分析法，该方法基于使用重组贾第虫InsP(6)激酶将InsP(6)与[³²P]-ATP磷酸化生成5-(PP)InsP(5)，并通过带有内部[³H]-(PP)InsP(5)标准品的阴离子交换高效液相色谱法对放射性标记的5-³²PInsP(5)产物进行定量。通过对添加已知量InsP(6)的相同组织等分试样进行测定，校正组织提取物中其他因素对酶反应的干扰。该分析法仅测定InsP(6)（而非其他肌醇磷酸），尽管其原理简单且无需专用或特殊设备，但耗时较长。不过，该分析法明确无误，能够准确量化细胞提取物中低至10飞摩尔的InsP(6)。
