使用磷酸酪氨酸对蛋白质酪氨酸磷酸酶进行连续分光光度测定。

Continuous spectrophotometric assay of protein tyrosine phosphatase using phosphotyrosine.

作者信息

Zhao Z, Zander N F, Malencik D A, Anderson S R, Fischer E H

机构信息

Department of Biochemistry, University of Washington, Seattle 98195.

出版信息

Anal Biochem. 1992 May 1;202(2):361-6. doi: 10.1016/0003-2697(92)90119-r.

DOI:10.1016/0003-2697(92)90119-r

PMID:1381565

Abstract

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.

摘要

已开发出一种以磷酸酪氨酸（P-Tyr）为底物的蛋白酪氨酸磷酸酶（PTP）连续活性测定法，并将其应用于测量两种纯化酶的活性，即全长T细胞蛋白酪氨酸磷酸酶（TC PTP）及其截短形式（TC delta C11 PTP）。通过P-Tyr去磷酸化引起的紫外线吸收和荧光变化来跟踪反应。两种酶均符合米氏动力学，TC PTP的Km = 304 microM，Vmax = 62,000单位/毫克，TC delta C11 PTP的Km = 194 microM，Vmax = 73,000单位/毫克。P-Tyr的D型和L型作为底物同样有效。两种酶的最适pH均为4.75。已知的PTP效应物对催化活性有预期的影响。