NONRADIOACTIVE METHOD TO MEASURE CD45 PROTEIN-TYROSINE-PHOSPHATASE ACTIVITY ISOLATED DIRECTLY FROM CELLS

Preparation of radioactive phosphorylated substrates is laborious, yie lds a limited amount of substrate with a short half-life and generates a low percentage of phosphorylated product which then has to be separ ated from non-phosphorylated material. These factors limit the usefuln ess of radioactive phosphorylated substrates in phosphatase assays and prohibit their use for kinetic analysis, which often requires large a mounts 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 chemica lly 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 a nd soluble phosphatases immunoprecipitated directly from cells. This c olorimetric microassay is performed in 96 well microtitre plates and c an reliably detect 100 pmol of free phosphate released, using a standa rd microplate reader. The phosphatase activity of CD45, a transmembran e PTP, was determined from as few as 1 X 10(4) lymphoid cells. The dev elopment of this colorimetric assay to measure immunoprecipitated CD45 PTP activity isolated from very small numbers of cells has general ap plicability for other PTPs and will help identify the cellular situati ons and conditions that result in changes in PTP activity.