Analysis of the metabolic turnover of the individual phosphate groups of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. Validation of novel analytical techniques by using 32P-labelled lipids from erythrocytes.
Analysis of the metabolic turnover of the individual phosphate groups of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. Validation of novel analytical techniques by using 32P-labelled lipids from erythrocytes.
复制标题
分析磷脂酰肌醇 4-磷酸和磷脂酰肌醇 4,5-二磷酸的各个磷酸基团的代谢周转。
作者：
P. Hawkins;R. Michell;C. Kirk
我们已经开发出的方法，产生估计的32 P含量的每个单独的磷酸基团的磷脂酰肌醇4-磷酸和磷脂酰肌醇4，5-二磷酸，从而扩展了信息，这些脂质在完整的细胞或膜制剂的标记的研究。用脱酰脂质进行分析。磷脂酰肌醇4，5-二磷酸的5-磷酸的测定通过在一级动力学条件下使用存在于分离的红细胞膜中的5-磷酸特异性磷酸单酯酶来实现[Downes，Mussat & Michell（1982）Biochem.J.203，169-177]。磷脂酰肌醇4-磷酸的4-磷酸和磷脂酰肌醇4，5-二磷酸的总磷酸单酯含量（4-磷酸加5-磷酸）的测定采用牛肠的碱性磷酸酶。1-磷酸盐的放射性是在碱性磷酸酶彻底处理后以有机磷酸盐形式残留的放射性。该方法已通过使用来自人红细胞的脂质进行了验证：这些脂质在其1-磷酸盐中不含32 P。这些方法应该是大量的价值在许多细胞的研究，显示快速激素扰动磷脂酰肌醇4，5-二磷酸代谢。
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.