Catalytic domains of the LAR and CD45 protein tyrosine phosphatases from Escherichia coli expression systems: purification and characterization for specificity and mechanism
| Autor: | Paul Burn, Shawn E. Ramer, Willi Bannwarth, Haruo Saito, Hyeongjin Cho, Michiyasu Itoh, Eric A. Kitas, Christopher T. Walsh |
|---|---|
| Rok vydání: | 1992 |
| Předmět: |
Cytoplasm
Stereochemistry Genetic Vectors Molecular Sequence Data Phosphatase Peptide Protein tyrosine phosphatase Biochemistry Catalysis Substrate Specificity Antigens CD HLA Antigens Histocompatibility Antigens Escherichia coli Humans Amino Acid Sequence Tyrosine Phosphotyrosine Gel electrophoresis chemistry.chemical_classification Membrane Glycoproteins biology Chemistry Active site Protein-Tyrosine Kinases Phosphorus-32 Recombinant Proteins Enzyme biology.protein Leukocyte Common Antigens |
| Zdroj: | Biochemistry. 31:133-138 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi00116a019 |
| Popis: | 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{sub cat}/K{sub M} value for a phosphotyrosyl undecapeptide. Sufficient purified LAR-D1 and LAR-D1D2 PTPases were available to demonstrate enzymatic exchange of {sup 18}O from {sup 18}O{sub 4} inorganic phosphate into H{sub 2} {sup 16}O at rates of {approximately}1 {times} 10{sup {minus}2} s{sup {minus}1}. The oxygen-18 exchange probably proceeds via a phosphoenzyme intermediate. Brief incubation of all three PTPase fragments with a ({sup 32}P)phosphotyrosyl peptide substrate prior to quench with SDS sample buffer and gel electrophoresis led to autoradiographic detection of {sup 32}P-labeled enzymes. Pulse/chase studies on the LAR {sup 32}P-enzyme showed turnover of the labeled phosphoryl group. |
| Databáze: | OpenAIRE |
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