PURIFICATION AND CHARACTERIZATION OF A SOLUBLE CATALYTIC FRAGMENT OF THE HUMAN TRANSMEMBRANE LEUKOCYTE ANTIGEN RELATED (LAR) PROTEIN TYROSINE PHOSPHATASE FROM AN ESCHERICHIA-COLI EXPRESSION SYSTEM

A 350 amino acid soluble fragment of the intracellular catalytic domain of the human transmembrane leukocyte antigen related (LAR) protein tyrosine phosphatase has been purified 17-fold to > 90% purity from an Escherichia coli expression vector in quantities sufficient for kinetic and structural characterization. To assess substrate specificity, phosphotyrosine peptides corresponding to autophosphorylation sites of the two major classes of tyrosine kinases have been synthesized. Thus 6-12-residue phosphotyrosine peptides of the insulin receptor and epidermal growth factor receptor kinase domains and of the autophosphorylation and C-terminal regulatory sites of p60src and p56lck have been analyzed for k(cat) and K(M) by using a nonradioactive chromogenic assay for P(i) release. The catalytic domain of LAR PTPase shows k(cat) values of 20-70 s-1 for phosphotyrosine peptides and affinities that vary 150-fold from 27-mu-M to 4.1 mM.