Measuring the specific activity of the CD45 protein tyrosine phosphatase.
Many assays for protein tyrosine phosphatases (PTPs) rely on colorimetric substrates or more recently developed fluorescent substrates. Enzyme activity is measured but no assessment of specific activity is made, which is important since these enzymes can exist in an enzymatically inactive form. In this study, we have directly compared the relative sensitivity of assays using either colorimetric or fluorescent substrates and show the latter to be considerably more sensitive with as few as 0.2 x 10(6) cells required compared to 3 x 10(6) cells for the colorimetric method. We then describe a quick and sensitive protocol that measures both the phosphatase activity and amount of CD45, allowing the calculation of the specific activity. CD45 is captured from cell lysates using anti-CD45 monoclonal antibody coated onto a 96-well plate, and the phosphatase activity is measured using the substrate fluorescein diphosphate (FDP). The amount of CD45 protein bound in the wells is measured against a standard curve using an anti-CD45-HRP conjugate, and this value is used to derive the specific activity. We used this assay to demonstrate that exposure of Jurkat T cells, and primary CD4(+) T cells to H(2)O(2) decreases the specific activity of their CD45, mimicking the changes seen in some diseases. The assay is applicable to other cell types and phosphatases, and so its use may help to identify the presence of inactive but intact enzyme in cells, which may play an important regulatory role in vivo.
Rider
David A
DA
Department of Rheumatology, Division of Immunity and Infection, School of Medicine, University of Birmingham, B15 2TT, Birmingham, UK.
Young
Stephen P
SP
D003124:Colorimetry / Q000379:methods; D019793:Fluorescein / Q000378:metabolism; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D019169:Jurkat Cells; D017493:Leukocyte Common Antigens / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*
D006861:Hydrogen Peroxide; D017493:Leukocyte Common Antigens; D017027:Protein Tyrosine Phosphatases; D019793:Fluorescein