A Continuous Spectrophotometric and Fluorometric Assay for Protein Tyrosine Phosphatase Using Phosphotyrosine-Containing Peptides

✍ Scribed by Z.Y. Zhang; D. Maclean; A.M. Thiemesefler; R.W. Roeske; J.E. Dixon

• Publisher: Elsevier Science
• Year: 1993
• Tongue: English
• Weight: 741 KB
• Volume: 211
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1993.1224

✦ Synopsis

Two continuous assays for protein tyrosine phosphatases (PTPases) have been developed using phosphotyrosine containing peptide substrates. These assays are based on the marked differences in the spectra of the peptide before and after the removal of the phosphate group. The increase in the absorbance at (282 \mathrm{~nm}) or the fluorescence at (305 \mathrm{~nm}) of the peptide upon the action of PTPase can be followed continuously and the resulting progress curve (time course) can be analyzed directly using the integrated form of the Michaelis-Menten equation. The procedure is convenient and efficient, since both (\boldsymbol{k}{\text {cat }}) and (K{m}) values can be obtained in a single run. The difference absorption coefficient ((\Delta \epsilon)) at 282 (\mathrm{nm}) is relatively insensitive to the (\mathrm{pH}) of the reaction media. These techniques were applied to two homogeneous recombinant PTPases employing six phosphotyrosine-containing peptides. (K_{m}) and (\boldsymbol{k}{\mathrm{cat}}) values obtained from the progress curve analysis were similar to those determined by the traditional initial rate inorganic phosphate assay. The peptides corresponding to autophosphorylation sites in Neu, (\mathbf{p 6}^{\text {lek }}), and (\mathbf{6 6 0 ^ { \text { erc } }}) proteins show distinct behavior with the Yersinia PTPase, Yop51*, and the mammalian PTPase (PTP1U323). In both cases, the (\boldsymbol{k}{\mathrm{cat}}) values were relatively constant for all the peptides tested whereas the (K_{m}) values were very sensitive to the amino acid sequence surrounding the tyrosine residue, especially in the case of Yop51*. Thus, both Yop51* and PTP1U323 show differential recognition of the phosphotyrosyl residues in the context of distinct primary structure of peptide substrates. 1983 Academic Press, Inc.