Cytosolic protein phosphotyrosine phosphatases from rabbit kidney. Purification of two distinct enzymes that bind to Zn2+-iminodiacetate agarose.
Cytosolic protein phosphotyrosine (PPT) phosphatase was measured using a new substrate, Tyr(32P)-labeled bovine serum albumin. Kidney was found as a particularly rich tissue source of PPT-phosphatase activity, containing twice as much as liver and over 10-fold more than brain, heart, lung, or skeletal muscle. An affinity column of Zn2+-iminodiacetate agarose adsorbed up to 60% of the PPT-phosphatase present in kidney extracts. Subsequent chromatography on DEAE-Sepharose separated the phosphatase into two peaks, labeled I and II, that had Mr = 34,000 and 37,000, respectively, upon gel filtration with Sephadex G-75 Superfine. Overall purification of 850- and 1100-fold was achieved with a net 4% yield. Both phosphatases hydrolyzed p-nitrophenylphosphate as well as the protein substrate in the presence of EDTA. Peak I phosphatase activity displayed a neutral pH optimum, had an absolute requirement for sulfhydryl compounds, and was sensitive to trypsin, whereas Peak II activity had an acidic pH optimum and was active without mercaptans. The two proteins also gave different fragmentation patterns by gel electrophoresis after digestion with S. aureus V8 protease. The results show that multiple forms of PPT phosphatase specifically interact with Zn2+ and provide a basis for further structural and functional comparisons among different members of the phosphoprotein phosphatase family.
D009597:4-Nitrophenylphosphatase / Q000032:analysis; D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007098:Imino Acids / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology*; D010749:Phosphoprotein Phosphatases / Q000302:isolation & purification* / Q000378:metabolism; D010766:Phosphorylation; D011817:Rabbits; D012685:Sepharose / Q000031:analogs & derivatives* / Q000378:metabolism
D007098:Imino Acids; C042660:zinc(II)-iminodiacetate agarose; D012685:Sepharose; D010749:Phosphoprotein Phosphatases; D009597:4-Nitrophenylphosphatase