A Continuous Spectrophotometric and Fluorometric Assay for Protein Tyrosine Phosphatase Using Phosphotyrosine-Containing Peptides
نویسندگان
چکیده
منابع مشابه
A continuous spectrophotometric assay for mitogen-activated protein kinase kinases.
We describe a convenient and simple continuous spectrophotometric method for the determination of mitogen-activated protein kinase (MAPK) kinase activity with its protein substrate. The assay relies on the measurement of phosphoprotein product generated in the first step of the MAPK kinase reaction. Dephosphorylation of the phosphoprotein is coupled to a MAPK phosphatase to generate phosphate, ...
متن کاملCells of Escherichia coli contain a protein-tyrosine kinase, Wzc, and a phosphotyrosine-protein phosphatase, Wzb.
Two proteins of Escherichia coli, termed Wzc and Wzb, were analyzed for their capacity to participate in the reversible phosphorylation of proteins on tyrosine. First, Wzc was overproduced from its specific gene and purified to homogeneity by affinity chromatography. Upon incubation in the presence of radioactive ATP, it was found to effectively autophosphorylate. Two-dimensional analysis of it...
متن کاملA phosphotyrosine-containing quenched fluorogenic peptide as a novel substrate for protein tyrosine phosphatases.
Mca-Gly-Asp-Ala-Glu-Tyr(PO(3)H(2))-Ala- Ala-Lys(DNP)-Arg-NH(2), where Mca is (7-methoxycoumarin-4-yl)acetyl and DNP is 2,4-dinitrophenyl, was synthesized as a fluorogenic substrate for protein tyrosine phosphatases (PTPs). In the peptide, the fluorescent Mca group is quenched efficiently by the DNP group. Although the fluorescence intensity of the substrate was practically unchanged upon PTP-ca...
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We report on the development of a sensitive real-time assay for monitoring the activity of L-asparaginase that hydrolyzes L-asparagine to L-aspartate and ammonia. In this method, L-aspartate is oxidized by L-aspartate oxidase to iminoaspartate and hydrogen peroxide (H2O2), and in the detection step horseradish peroxidase uses H2O2 to convert the colorless, nonfluorescent reagent Amplex Red to t...
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A continuous spectrophotometric assay has been developed for detecting β-glucuronidase activity. In the assay, Para-nitrophenyl β-D-glucuronide is cleaved to yield a chromophoric product. With the commercial E. coli enzyme, it is demonstrated that the reactions can be continuously monitored by the increase of absorbance at 405 nm. The method is highly sensitive and able to detect less than 1.4 ...
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ژورنال
عنوان ژورنال: Analytical Biochemistry
سال: 1993
ISSN: 0003-2697
DOI: 10.1006/abio.1993.1224