Hawkins P T, Michell R H, Kirk C J
Biochem J. 1984 Mar 15;218(3):785-93. doi: 10.1042/bj2180785.
We have developed methods that yield estimates of the 32P content of each of the individual phosphate groups of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, thus extending the information available from studies of the labelling of these lipids in intact cells or membrane preparations. The analyses are undertaken with the deacylated lipids. Assay of the 5-phosphate of phosphatidylinositol 4,5-bisphosphate is achieved by the use, under conditions of first-order kinetics, of a 5-phosphate-specific phosphomonoesterase present in isolated erythrocyte membranes [Downes, Mussat & Michell (1982) Biochem. J. 203, 169-177]. Assay of the 4-phosphate of phosphatidylinositol 4-phosphate and of the total monoester phosphate content (4-phosphate plus 5-phosphate) of phosphatidylinositol 4,5-bisphosphate employs alkaline phosphatase from bovine intestine. The radioactivity of the 1-phosphate is that remaining as organic phosphate after exhaustive alkaline phosphatase treatment. The methodology has been validated by using lipids from human erythrocytes: these contain no 32P in their 1-phosphate. These methods should be of substantial value in studies of the many cells that show rapid hormonal perturbations of phosphatidylinositol 4,5-bisphosphate metabolism.
我们已经开发出一些方法，能够估算磷脂酰肌醇4-磷酸和磷脂酰肌醇4,5-二磷酸中各个磷酸基团的32P含量，从而扩展了在完整细胞或膜制剂中对这些脂质标记研究可得的信息。分析是对脱酰基脂质进行的。在一级动力学条件下，通过使用分离的红细胞膜中存在的5-磷酸特异性磷酸单酯酶来测定磷脂酰肌醇4,5-二磷酸的5-磷酸[唐斯、穆萨特和米切尔（1982年）《生物化学杂志》203卷，第169 - 177页]。用牛小肠碱性磷酸酶来测定磷脂酰肌醇4-磷酸的4-磷酸以及磷脂酰肌醇4,5-二磷酸的总单酯磷酸含量（4-磷酸加5-磷酸）。1-磷酸的放射性是经过彻底碱性磷酸酶处理后作为有机磷酸残留的放射性。该方法已通过使用来自人红细胞的脂质得到验证：这些脂质的1-磷酸中不含32P。这些方法在研究许多显示磷脂酰肌醇4,5-二磷酸代谢快速激素扰动的细胞方面应具有重要价值。
