Ng D H, Harder K W, Clark-Lewis I, Jirik F, Johnson P
Biomedical Research Center, University of British Columbia, Vancouver, Canada.
J Immunol Methods. 1995 Feb 27;179(2):177-85. doi: 10.1016/0022-1759(94)00281-z.
Preparation of radioactive phosphorylated substrates is laborious, yields a limited amount of substrate with a short half-life and generates a low percentage of phosphorylated product which then has to be separated from non-phosphorylated material. These factors limit the usefulness of radioactive phosphorylated substrates in phosphatase assays and prohibit their use for kinetic analysis, which often requires large amounts of substrate. An alternative method for the kinetic analysis of purified or recombinant soluble phosphatases uses the malachite green reagent which can detect nanomoles of phosphate released from chemically synthesized phosphorylated peptides. In this report we describe a rapid and sensitive non-radioactive method that can be used to measure protein tyrosine phosphatase (PTP) activities of both transmembrane and soluble phosphatases immunoprecipitated directly from cells. This colorimetric microassay is performed in 96 well microtitre plates and can reliably detect 100 pmol of free phosphate released, using a standard microplate reader. The phosphatase activity of CD45, a transmembrane PTP, was determined from as few as 1 x 10(4) lymphoid cells. The development of this colorimetric assay to measure immunoprecipitated CD45 PTP activity isolated from very small numbers of cells has general applicability for other PTPs and will help identify the cellular situations and conditions that result in changes in PTP activity.
放射性磷酸化底物的制备过程繁琐，底物产量有限且半衰期短，磷酸化产物的生成比例低，随后还必须将其与未磷酸化的物质分离。这些因素限制了放射性磷酸化底物在磷酸酶测定中的实用性，并使其无法用于动力学分析，因为动力学分析通常需要大量底物。一种用于纯化或重组可溶性磷酸酶动力学分析的替代方法是使用孔雀石绿试剂，该试剂可检测从化学合成的磷酸化肽中释放的纳摩尔磷酸盐。在本报告中，我们描述了一种快速灵敏的非放射性方法，可用于测量直接从细胞中免疫沉淀的跨膜和可溶性磷酸酶的蛋白酪氨酸磷酸酶（PTP）活性。这种比色微量测定法在96孔微量滴定板中进行，使用标准酶标仪可可靠地检测到释放的100皮摩尔游离磷酸盐。跨膜PTP CD45的磷酸酶活性可从低至1×10⁴个淋巴细胞中测定。这种用于测量从极少量细胞中分离出的免疫沉淀CD45 PTP活性的比色测定法的开发，对其他PTP具有普遍适用性，并将有助于确定导致PTP活性变化的细胞情况和条件。
