Dhw. Ng et al., NONRADIOACTIVE METHOD TO MEASURE CD45 PROTEIN-TYROSINE-PHOSPHATASE ACTIVITY ISOLATED DIRECTLY FROM CELLS, Journal of immunological methods, 179(2), 1995, pp. 177-185
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.