Expression, purification, and characterization of SH2-containing protein tyrosine phosphatase, SH-PTP2
| Autor: | S, Sugimoto, R J, Lechleider, S E, Shoelson, B G, Neel, C T, Walsh |
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| Rok vydání: | 1993 |
| Předmět: |
Phosphopeptides
Molecular Sequence Data Hydrogen-Ion Concentration Chromatography Ion Exchange Peptide Fragments Recombinant Proteins Substrate Specificity ErbB Receptors Molecular Weight Kinetics Escherichia coli Humans Thermodynamics Electrophoresis Polyacrylamide Gel Receptors Platelet-Derived Growth Factor Amino Acid Sequence Cloning Molecular Protein Tyrosine Phosphatases |
| Zdroj: | The Journal of biological chemistry. 268(30) |
| ISSN: | 0021-9258 |
| Popis: | A human protein tyrosine phosphatase containing two src homology 2 (SH2) domains (SH-PTP2) was expressed in Escherichia coli under T7 promoter control and purified to near homogeneity. The purified protein, with molecular mass of 68 kDa on SDS-polyacrylamide gel electrophoresis, was identified as SH-PTP2 by its protein tyrosine phosphatase activity and N-terminal amino acid sequence analysis. Its protein tyrosine phosphatase activity was sensitive to pH and salt concentration. Whereas its optimum pH for the low molecular weight substrate para-nitrophenyl phosphate is 5.6, the pH optima for peptide substrates were shifted toward neutral. With the artificial protein substrate reduced, carboxyamidomethylated, and maleylated lysozyme, it displays 2000-fold lower Km (1.7 microM) and 2.4-fold higher kcat (0.11 s-1) than with para-nitrophenyl phosphate. Among the phosphopeptides from autophosphorylation sites of receptors for epidermal growth factor and platelet-derived growth factor, SH-PTP2 displayed high activity toward phosphopeptides corresponding to pY992 of the epidermal growth factor receptor and pY1009 and pY1021 of the platelet-derived growth factor receptor. In further enzymatic studies with phosphopeptides corresponding to pY1009, SH-PTP2 showed nonlinear Line-weaver-Burk double-reciprocal plots, suggesting that the phosphopeptide corresponding to pY1009 may have a substrate and allosteric effect. |
| Databáze: | OpenAIRE |
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