Continuous spectrophotometric assay of protein tyrosine phosphatase using phosphotyrosine.
A continuous activity assay for protein tyrosine phosphatases (PTPs), employing phosphotyrosine (P-Tyr) as a substrate, has been developed and applied to measure the activities of two purified enzymes, namely, the full length T-cell protein tyrosine phosphatase (TC PTP) and its truncated form (TC delta C11 PTP). The reaction was followed by changes in ultraviolet absorption and fluorescence resulting from the dephosphorylation of P-Tyr. Both enzymes obey Michaelis-Menten kinetics, with Km = 304 microM, Vmax = 62,000 units/mg for TC PTP and Km = 194 microM, Vmax = 73,000 units/mg for TC delta C11 PTP. The D- and L-forms of P-Tyr are equally effective as substrates. The optimum pH for both enzymes is 4.75. The known effectors of PTPs have the predicted effects on catalytic activity.
Zhao
Z
Z
Department of Biochemistry, University of Washington, Seattle 98195.
Zander
N F
NF
Malencik
D A
DA
Anderson
S R
SR
Fischer
E H
EH
D006863:Hydrogen-Ion Concentration; D019000:Phosphotyrosine; D017027:Protein Tyrosine Phosphatases / Q000032:analysis* / Q000493:pharmacokinetics; D013053:Spectrophotometry / Q000379:methods; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry
D019000:Phosphotyrosine; D014443:Tyrosine; D017027:Protein Tyrosine Phosphatases