Multiple forms of phosphotyrosyl- and phosphoseryl-protein phosphatase from cardiac muscle: partial purification and characterization of an EDTA-stimulated phosphotyrosyl-protein phosphatase.
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 Mr = 65,000 (Stokes radius = 3.8 nm; S020,W = 4.1). Its activity was stimulated by 5- to 10-fold in the presence of EDTA (Ka = 15 microM) 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.
D000818:Animals; D001921:Brain / Q000201:enzymology; D002614:Chelating Agents / Q000494:pharmacology; D002848:Chromatography, DEAE-Cellulose; D004492:Edetic Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000302:isolation & purification*; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D010749:Phosphoprotein Phosphatases / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
D002614:Chelating Agents; D007527:Isoenzymes; D004492:Edetic Acid; D010749:Phosphoprotein Phosphatases