DEPHOSPHORYLATION OF TYROSINE-PHOSPHORYLATED SYNTHETIC PEPTIDES BY RAT-LIVER PHOSPHOTYROSINE PROTEIN PHOSPHATASE ISOENZYMES

Five phosphotyrosine-containing peptides have been synthesized by FMOC solid-phase peptide synthesis. These peptides correspond to the 411-4 19 sequence of the Xenopus src oncogene, to the 1191-1220 sequence of the human EGF receptor precursor, to the 1146-1158 sequence of the hum an insulin receptor, to the 856-865 sequence of the human beta-PDGF re ceptor, and to the 5-16 sequence of the erythrocyte human band 3. The peptides were used as substrates for activity assay of two isoforms (A cP1 and AcP2) of a low molecular weight cytosolic PTPase. The assay, p erformed in microtiter EIA plates using Malachite green to determine t he released phosphate, was rapid, reproducible, and sensitive. Both PT Pase isoforms were able to hydrolyze all synthesized peptides, though with different affinity and rate. The main kinetic parameters were com pared and discussed with respect to the role of the two enzymes in the cell.