Fluorometric and time-resolved immunofluorometric assays for protein-tyrosine phosphatase activity.
OBJECTIVE
To develop sensitive nonisotopic assays for protein-tyrosine phosphatase (PTP) activity.


METHODS
The fluorometric assay is based on the fact that phosphotyrosine but not tyrosine forms highly fluorescent complexes with Tb3+. Thus, PTP activity can be followed by measuring the decrease of fluorescence due to hydrolysis of phosphotyrosine. The time-resolved immunofluorometric assay employs tyrosine-phosphorylated substrates, immobilized on microtitre wells. After incubation with PTP, the remaining phosphotyrosine residues are reacted with an antiphosphotyrosine antibody. The immunocomplexes formed are detected with an alkaline phosphatase (ALP)-labeled second antibody. The phosphate ester of 5' fluorosalicylate (FSAP) is used as substrate. The fluorosalicylate produced forms highly fluorescent complexes with Tb3+ - EDTA in alkaline solution. The fluorescence is measured with a time-resolved fluorometer.


RESULTS
The truncated form of the T-cell protein tyrosine phosphatase (TCdeltaC11 PTP) was determined in the range 1100-36,500 U/L by the fluorometric assay and 36-7100 U/L by the time-resolved immunofluorometric assay.


CONCLUSIONS
The two nonisotopic assays should prove beneficial for the determination and study of various PTP.
Galvan
B
B
Department of Chemistry and Biochemistry, University of Windsor, Ontario, Canada.
Christopoulos
T K
TK
D005069:Evaluation Studies as Topic; D015200:Fluoroimmunoassay / Q000379:methods* / Q000706:statistics & numerical data; D005470:Fluorometry / Q000379:methods* / Q000706:statistics & numerical data; D006801:Humans; D006863:Hydrogen-Ion Concentration; D017027:Protein Tyrosine Phosphatases / Q000032:analysis*; D012680:Sensitivity and Specificity; D013601:T-Lymphocytes / Q000201:enzymology; D013997:Time Factors
D017027:Protein Tyrosine Phosphatases