title: A general peptide substrate for protein tyrosine phosphatases.
abstract: The determination of protein tyrosine phosphatase (PTP) activity using different protein substrates such as modified lysozyme or myelin basic protein is greatly affected by the type of enzyme involved, the condition of the assay, and the presence of effectors. The purpose of this study was to develop a general substrate of wide applicability with which variations in enzymatic activity would be minimized. A nonapeptide ENDYINASL derived from a highly conserved region of the T-cell phosphatase TC.PTP (Cool et al. (1989) Proc. Natl. Acad. Sci. USA 86, 5257-5261) was phosphorylated with a recombinant tyrosine kinase domain of the EGF receptor and purified on a C18 cartridge. Phosphatase activities of intracellular and receptor-linked PTPs were as high as the highest values obtained with the protein substrates. The intracellular, low-molecular-weight PTPs exhibited Km values between 0.5 and 1.3 microM, whereas the receptor forms CD45 and RPTP alpha gave values of 14 and 35 microM, respectively. All PTPs displayed similar properties toward the peptide including a low pH optimum and inhibition by vanadate, divalent cations, and heparin. Following immunoprecipitation, 1 ng of TC.PTP could be detected with ENDY(P)INASL compared to 10 ng in presence of protein substrates.
authors.affiliation: Department of Biochemistry, University of Washington, Seattle 98195.
authors.forename: G
authors.initials: G
authors.lastname: Daum
authors.forename: F
authors.initials: F
authors.lastname: Solca
authors.forename: C D
authors.initials: CD
authors.lastname: Diltz
authors.forename: Z
authors.initials: Z
authors.lastname: Zhao
authors.forename: D E
authors.initials: DE
authors.lastname: Cool
authors.forename: E H
authors.initials: EH
authors.lastname: Fischer
mesh_terms: D000595:Amino Acid Sequence; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D009842:Oligopeptides; C081990:glutamyl-asparaginyl-aspartyl-tyrosyl-isoleucyl-asparaginyl-alanyl-seryl-leucine; D017027:Protein Tyrosine Phosphatases