Continuous and Discontinuous Assays for Phosphotyrosyl Protein Phosphatase Activity Using Phosphotyrosyl Peptide Substrates

Abstract Assays for phosphotyrosyl protein phosphatase activity have been developed using the synthetic peptide Glu-Glu-Tyr(P)-Ala-Ala and its N α -fluorenylme-thoxycarbonyl derivative (Fmoc-Glu-Glu-Tyr(P)-Ala-Ala) as substrates. Conditions for monitoring the phosphatase-catalyzed hydrolysis of the former pep-tide, either spectrophotometrically or fluorometrically, are reported. These continuous assays are similar in sensitivity to published assays using phosphotyrosine as substrate. A discontinuous HPLC-based assay using the intensely fluorescent Fmoc-peptide as substrate is also described. This assay is comparable in sensitivity to assays using 32 P-labeled substrates and is suitable for assaying low levels of phosphotyrosyl protein phosphatase activity in crude tissue extracts.