CATALYTIC DOMAINS OF THE LAR AND CD45 PROTEIN TYROSINE PHOSPHATASES FROM ESCHERICHIA-COLI EXPRESSION SYSTEMS - PURIFICATION AND CHARACTERIZATION FOR SPECIFICITY AND MECHANISM

The cytoplasmic domains of two human transmembrane protein tyrosine phosphatases (PTPases), LAR and CD45, have been expressed in Escherichia coli, purified to near-homogeneity, and compared for catalytic efficiency toward several phosphotyrosine-containing peptide substrates. A 615-residue LAR fragment (LAR-D1D2) containing both tandemly repeated PTPase domains shows almost identical specific activity and high catalytic efficiency as the 40-kDa single-domain LAR-D1 fragment, consistent with a single functional active site in the 70-kDa LAR-D1D2 enzyme. A 90-kDa fragment of the human leukocyte CD45 PTPase, containing two similar tandemly repeated PTPase domains, shows parallel specificity to LAR-D1 and LAR-D1D2 with a high k(cat)/K(m) value for a phosphotyrosyl undecapeptide. Sufficient purified LAR-D1 and LAR-D1D2 PTPases were available to demonstrate enzymatic exchange of O-18 from O-18(4) inorganic phosphate into (H2O)-O-16 at rates of approximately 1 X 10(-2) s-1. The oxygen-18 exchange probably proceeds via a phosphoenzyme intermediate. Brief incubation of all three PTPase fragments with a [P-32]phosphotyrosyl peptide substrate prior to quench with SDS sample buffer and gel electrophoresis led to autoradiographic detection of P-32-labeled enzymes. Pulse/chase studies on the LAR P-32-enzyme showed turnover of the labeled phosphoryl group.