Partial purification and characterization of phosphotyrosyl-protein phosphatase from Ehrlich ascites tumor cells

Abstract

A phosphotyrosyl-protein phosphatase in membrane vesicles from human epidermoid carcinoma A431 cells was previously described which is inhibited by micromolar concentrations of Zn2+ and is insensitive to EDTA and NaF. The identification and partial purification of a similar enzyme from lysates of [mouse] Ehrlich ascites tumor cells is presented. The enzyme was purified by using DEAE Sephadex, Zn2+ affinity, and Sephadex G-75 chromatography. During purification, the phosphatase was separated into at least 3 fractions, all of which exhibited very similar properties and an apparent MW of 40,000 upon gel filtration. The enzyme dephosphorylated phosphotyrosine (P-Tyr)-containing carboxymethylated and succinylated (CM-SC) phosphorylase with an apparent Km of 0.8 .mu.M, as well as P-Tyr-containing casein and epidermal growth factor (EGF) receptor kinase, but did not dephosphorylate P-Ser-phosphorylase. The phosphatase was inhibited by Zn2+ at micromolar concentrations (K0.5 with EGF receptor kinase = 5 .times. 10-6 M; with CM-SC-phosphorylase = 3.3 .times. 10-5 M) but not by millimolar concentrations of EDTA and NaF. No inhibition was seen with 1 mM tetramisole, a specific inhibitor of alkaline phosphatases. P-Tyr inhibited the enzyme by 50% at 0.4 .times. 10-3 M, while Tyr, Pi, PPi, and p-nitrophenyl phosphate, an excellent substrate for alkaline phosphatases and structurally very similar to P-Tyr, exerted partial inhibition at concentrations above 10-3 M. The pH optimum was 6.5-7, depending on the substrate used. Very little activity was seen below pH 5 and above pH 8.5. These properties clearly distinguish this enzyme from alkaline phosphatases, as well as the neutral and acidic protein phosphatases so far described, and therefore define it as a new enzyme of the phosphatase family, a phosphotyrosyl-protein phosphatase.