Two nonradioactive assays for phosphotyrosine phosphatases with activity toward the insulin receptor.
Two highly sensitive, nonradiolabeled assays for protein phosphotyrosine phosphatase (PTPase) have been developed. The first assay is based on the use of chemically synthesised phosphotyrosine-containing peptides that can be separated from the dephosphorylated peptide products by HPLC. In this assay, partially purified placental PTPase 1B dephosphorylated three dodecaphosphopeptides (corresponding to insulin receptor autophosphorylation sites at positions PY1146, PY1150, and PY1151) with approximately equal affinity (Km 1.3-2.5 microM), indicating that PTPase 1B shows no distinct preference for the site of dephosphorylation in these peptides. The second assay employs either a phosphopeptide or an autophosphorylated tyrosine kinase domain immobolized on microtiter plate wells. After reaction with PTPase, the remaining unconverted phosphosubstrate is detected in an ELISA using anti-phosphotyrosine antibodies. The latter assay was used to monitor PTPase activity during purification procedures and for characterizing PTPases. Modulation of PTPase activity by orthovanadate, heparin, Zn2+, and EDTA gave similar results in both assays. The immobilized autophosphorylated IR tyrosine kinase domain was a poor substrate for bovine liver alkaline phosphatase and seminal fluid acid phosphatase. The second assay also offers the potential for comparing PTPase activity toward several autophosphorylated tyrosine kinase domains, including those of the insulin, epidermal growth factor, and platelet-derived growth factor receptors.
Madden
J A
JA
Department of Biochemistry, Glaxo Group Research, Greenford, Middlesex, United Kingdom.
Bird
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Myles
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D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003619:Dansyl Compounds / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D010766:Phosphorylation; D010920:Placenta / Q000737:chemistry; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000032:analysis* / Q000187:drug effects; D011972:Receptor, Insulin / Q000378:metabolism*; D013379:Substrate Specificity; D014638:Vanadates / Q000494:pharmacology; D015032:Zinc / Q000494:pharmacology
D003619:Dansyl Compounds; D009842:Oligopeptides; D014638:Vanadates; D006493:Heparin; D011972:Receptor, Insulin; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases; D015032:Zinc