Continuous and discontinuous assays for phosphotyrosyl protein phosphatase activity using phosphotyrosyl peptide substrates.
Assays for phosphotyrosyl protein phosphatase activity have been developed using the synthetic peptide Glu-Glu-Tyr(P)-Ala-Ala and its N alpha-fluorenylmethoxycarbonyl derivative (Fmoc-Glu-Glu-Tyr(P)-Ala-Ala) as substrates. Conditions for monitoring the phosphatase-catalyzed hydrolysis of the former peptide, either spectrophotometrically or fluorometrically, are reported. These continuous assays are similar in sensitivity to published assays using phosphotyrosine as substrate. A discontinuous HPLC-based assay using the intensely fluorescent Fmoc-peptide as substrate is also described. This assay is comparable in sensitivity to assays using 32P-labeled substrates and is suitable for assaying low levels of phosphotyrosyl protein phosphatase activity in crude tissue extracts.
Nash
K
K
Department of Biochemistry, University of Queensland, St. Lucia, Brisbane, Australia.
Feldmuller
M
M
de Jersey
J
J
Alewood
P
P
Hamilton
S
S
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D005449:Fluorenes / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism*; D010748:Phosphopeptides / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014443:Tyrosine / Q000378:metabolism
D005449:Fluorenes; D009842:Oligopeptides; D010748:Phosphopeptides; C083915:glutamyl-glutamyl-phosphotyrosyl-alanyl-alanine; C083916:N-(alpha)fluorenylmethoxycarbonyl-glutamyl-glutamyl-phosphotyrosyl-alanyl-alanine; D014443:Tyrosine; D017027:Protein Tyrosine Phosphatases