Rivera J, Lozano M L, Gonzalez-Conejero R, Corral J, De Arriba F, Vicente V
Unit of Hematology and Hemotherapy, School of Medicine, Hospital General Universitario, Murcia, Spain.
Anal Biochem. 1998 Feb 1;256(1):117-21. doi: 10.1006/abio.1997.2488.
The binding of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] to saponin-permeabilized blood-bank-outdated human platelets, 6 days old, has been characterized (Kd = 3.8 nM; Bmax = 1.7 pmol/mg protein) and used to develop a novel radioreceptor assay which allows the measurement of the Ins(1,4,5)P3 content in resting or agonist-stimulated cells. This assay is as sensitive (0.25 pmol in a 0.25 ml volume), specific, and reproducible as previously proposed methods. In addition, obtaining large batches of the Ins(1,4,5)P3 binding protein by treating outdated platelets with saponin is simple and quick and uses otherwise discarded material. Moreover, the assay is considerably cheaper than commercially available kits. Using this method we confirmed that thrombin evokes a rapid, transient, and dose-dependent increase in the platelet concentration of Ins(1,4,5)P3.
已对肌醇1,4,5 - 三磷酸[Ins(1,4,5)P3]与皂素通透处理的6日龄血库过期人血小板的结合特性进行了表征（解离常数Kd = 3.8 nM；最大结合量Bmax = 1.7 pmol/mg蛋白质），并用于开发一种新型放射受体测定法，该方法可测量静息或激动剂刺激细胞中的Ins(1,4,5)P3含量。此测定法与先前提出的方法一样灵敏（在0.25 ml体积中为0.25 pmol）、特异且可重复。此外，通过用皂素处理过期血小板来大量获取Ins(1,4,5)P3结合蛋白既简单又快速，且利用了原本会被丢弃的材料。而且，该测定法比市售试剂盒便宜得多。使用此方法，我们证实凝血酶可引起血小板中Ins(1,4,5)P3浓度快速、短暂且剂量依赖性的增加。
