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 in hibition of membrane-bound CD45 from Jurkat cells. This assay is based on t he fact that similar to 64% of PTP activity from Jurkat cell membrane is co ntributed by CD45, This has been proven by comparing the activity in membra ne protein from wildtype Jurkat cells and CD45-negative mutant cells, and a lso by measuring the residual activity after depleting CD45 from Jurkat cel l membrane. We have demonstrated that fluorescein diphosphate can be used a s a substrate to monitor CD45 activity from Jurkat cell membrane, which all ows us to easily follow CD45 activity in both fluorescent and absorbance mo des in a 96-well format. Some common protein tyrosine phosphatase inhibitor s have been evaluated with this assay, (C) 2000 Academic Press.