Multiple forms of phosphotyrosyl- and phosphoseryl-protein phosphatase from cardiac muscle: Partial purification and characterization of an EDTA-stimulated phosphotyrosyl-protein phosphatase

• Published in: Archives of biochemistry and biophysics Vol. 226; no. 2; pp. 517 - 530
• Main Authors: Chernoff, Jonathan, Li, Heng-Chun
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.10.1983
• Subjects: Animals; Brain - enzymology; Chelating Agents - pharmacology; Chromatography, DEAE-Cellulose; Edetic Acid - pharmacology; Hydrogen-Ion Concentration; Isoenzymes - isolation & purification; Kinetics; Mice; Molecular Weight; Myocardium - enzymology; Phosphoprotein Phosphatases - isolation & purification; Phosphoprotein Phosphatases - metabolism; Substrate Specificity
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/0003-9861(83)90321-1

Chromatography of cardiac muscle and brain extracts on DEAE-cellulose resolved phosphotyrosyl-protein phosphatase activity into three fractions, termed Y-1, Y-2 and Y-3. These were eluted at 0.05, 0.15 and 0.3 m KCl, representing about 33, 55 and 12%, respectively, of the enzymatic activity recovered from the resin. Comparative studies demonstrated that the properties of phosphatases Y-1, Y-2 and Y-3 were distinctly different from those of previously identified phosphoseryl-protein phosphatases-1, -2, -3, and -4. Phosphatases Y-1, Y-2 and Y-3 were stimulated by EDTA and exhibited optimal activity at neutral pH. These properties were different from those of the two minor phosphotyrosyl-protein phosphatase activities associated with phosphoseryl-protein phosphatases-3, and -4, which were divalent cation dependent and exhibited optimal activity at alkaline pH. Further purification of phosphatase Y-2 from bovine heart has been carried out. The enzyme had a M r = 65,000 (Stokes radius = 3.8 nm; s 20,w 0 = 4.1). Its activity was stimulated by 5- to 10-fold in the presence of EDTA ( K a = 15 μM) and was strongly inhibited by micromolar concentrations of vanadate. Phosphatase Y-2 was highly specific for phosphotyrosyl-IgG and -casein, and showed little activity toward phosphoseryl-casein, -phosphorylase a, phosphothreonyl-inhibitor-1 and p-nitrophenyl phosphate. The present studies indicate that phosphotyrosyl-protein phosphatase activity in animal tissues exists in multiple forms. The major active species are specific for phosphotyrosyl proteins and represent enzymes different from the known phosphoseryl-protein phosphatases and p-nitrophenyl phosphatases.