title: Non-radioactive method to measure CD45 protein tyrosine phosphatase activity isolated directly from cells.
abstract: 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.
authors.affiliation: Biomedical Research Center, University of British Columbia, Vancouver, Canada.
authors.forename: D H
authors.initials: DH
authors.lastname: Ng
authors.forename: K W
authors.initials: KW
authors.lastname: Harder
authors.forename: I
authors.initials: I
authors.lastname: Clark-Lewis
authors.forename: F
authors.initials: F
authors.lastname: Jirik
authors.forename: P
authors.initials: P
authors.lastname: Johnson
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D017493:Leukocyte Common Antigens / Q000032:analysis*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D019000:Phosphotyrosine; D011233:Precipitin Tests; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D013379:Substrate Specificity; D014443:Tyrosine / Q000031:analogs & derivatives / Q000737:chemistry
chemical_list: D010455:Peptides; D019000:Phosphotyrosine; D014443:Tyrosine; D017493:Leukocyte Common Antigens; D017027:Protein Tyrosine Phosphatases