Purification of a specific reversible tyrosine-O-phosphate phosphatase.
A phosphatase specific for tyrosine-O-phosphate (Tyr-P) was separated from several nonspecific phosphatases present in the third instar larvae of Drosophila melanogaster. The enzyme hydrolyzed L-Tyr-P, with an apparent Km of 0.14 mM, but not D-Tyr-P after being freed from hydrolytic activity toward p-nitrophenyl phosphate, the common phosphatase substrate. Such purified preparations also catalyzed a reversible phosphate transfer reaction from unlabeled Tyr-P to [3H]tyrosine. The transfer activity was L4-14% of the hydrolytic activity, depending on the initial concentration of tyrosine (0.25-4.0 mM). The two activities coincided throughout purification. However, they differed in pH optimum, that of hydrolysis being 6.5-7 and that of phosphate transfer being 7.7.5. The two activities were also differentially inhibited by 1-p-bromotetramisole oxalate in the presence of EDTA and by Mn2+. Addition of Mg2+ did not affect either hydrolysis or phosphate transfer, but 5 mM Zn2+ was 65% inhibitory to both. Sodium fluoride strongly inhibited both reactions, and this inhibition was reversed by EDTA, while EDTA itself had no effect. Pi had no effect and no detectable incorporation of 32Pi into Tyr-P was observed, indicating that the phosphate transfer reaction is not a simple reversal of hydrolysis. No ATP-linked phosphorylation of tyrosine was found.
D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D004331:Drosophila melanogaster / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D019000:Phosphotyrosine; D013379:Substrate Specificity; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D002413:Cations, Divalent; D019000:Phosphotyrosine; D014443:Tyrosine; C023210:tyrosine phosphate hydrolase; D010744:Phosphoric Monoester Hydrolases