Continuous spectrophotometric assay of protein tyrosine phosphatase using phosphotyrosine

✍ Scribed by Zhizhuang Zhao; Norbert F. Zander; Dean A. Malencik; Sonia R. Anderson; Edmond H. Fischer

• Publisher: Elsevier Science
• Year: 1992
• Tongue: English
• Weight: 606 KB
• Volume: 202
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(92)90119-r

✦ Synopsis

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.