Development of a method for evaluating protein tyrosine phosphatase CD45 inhibitors using Jurkat cell membrane.
A simple, high-throughput fluorescent assay was developed to measure the inhibition of membrane-bound CD45 from Jurkat cells. This assay is based on the fact that approximately 64% of PTP activity from Jurkat cell membrane is contributed by CD45. This has been proven by comparing the activity in membrane protein from wild-type Jurkat cells and CD45-negative mutant cells, and also by measuring the residual activity after depleting CD45 from Jurkat cell membrane. We have demonstrated that fluorescein diphosphate can be used as a substrate to monitor CD45 activity from Jurkat cell membrane, which allows us to easily follow CD45 activity in both fluorescent and absorbance modes in a 96-well format. Some common protein tyrosine phosphatase inhibitors have been evaluated with this assay.
Waddleton
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Department of Biochemistry and Molecular Biology, Merck Frosst Center for Therapeutic Research, Pointe-Claire, Dorval, Quebec, H9R 4P8, Canada.
Ramachandran
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Wang
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D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D019169:Jurkat Cells; D017493:Leukocyte Common Antigens / Q000187:drug effects*; D013050:Spectrometry, Fluorescence / Q000379:methods*
D004791:Enzyme Inhibitors; D017493:Leukocyte Common Antigens