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 kcat/Km value for a phosphotyrosyl undecapeptide. Sufficient purified LAR-D1 and LAR-D1D2 PTPases were available to demonstrate enzymatic exchange of 18O from 18O4 inorganic phosphate into H2(16)O 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 [32P]phosphotyrosyl peptide substrate prior to quench with SDS sample buffer and gel electrophoresis led to autoradiographic detection of 32P-labeled enzymes. Pulse/chase studies on the LAR 32P-enzyme showed turnover of the labeled phosphoryl group.
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Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts.
Ramer
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Itoh
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Kitas
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Bannwarth
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Burn
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Saito
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Walsh
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D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D002384:Catalysis; D003593:Cytoplasm / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006680:HLA Antigens / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D006649:Histocompatibility Antigens / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D017493:Leukocyte Common Antigens; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D013379:Substrate Specificity; D014443:Tyrosine / Q000031:analogs & derivatives / Q000276:immunology / Q000378:metabolism
D015703:Antigens, CD; D006680:HLA Antigens; D006649:Histocompatibility Antigens; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D017493:Leukocyte Common Antigens