Purification and characterization of a protein-phosphotyrosine phosphatase from rat spleen which dephosphorylates and inactivates a tyrosine-specific protein kinase.
A particulate form of protein-phosphotyrosine phosphatase was solubilized and purified over 2,000-fold from the particulate fraction of rat spleen. Phosphorylated poly(Glu, Tyr), a random copolymer of glutamic acid and tyrosine, was used as substrate for measuring protein-phosphotyrosine phosphatase activity. Nonionic detergents like Triton X-100 increased the protein-phosphotyrosine phosphatase activity of the particulate fraction (but not of the soluble fraction) by 4-8-fold. Chromatography of the Triton extract of the particulate fraction on DEAE-Sephacel gave three peaks of protein-phosphotyrosine phosphatase activity. The major peak of activity was further purified on Bio-Gel HTP, Sephadex G-75, and phosphocellulose columns. On polyacrylamide gel electrophoresis in the presence of Na-dodecyl-SO4 the purified enzyme showed a major protein band of Mr 36,000 which comigrated with enzyme activity on the phosphocellulose column. The apparent Vmax and Km for phosphorylated poly(Glu,Tyr) were 6,150 nmol min-1 mg-1 and 1.6 microM, respectively. This enzyme was strongly inhibited by microM concentrations of orthovanadate and zinc acetate. Fluoride (50 mM) inhibited this enzyme only by 30-40%. Divalent metal ions Ca2+, Mg2+, and Mn2+ were inhibitory at 1-10 mM concentration. EDTA had no effect on the activity of the purified enzyme. This phosphatase could dephosphorylate and inactivate the phosphorylated form of a tyrosine-specific protein kinase (TK-I) previously purified from rat spleen. Dephosphorylation and inactivation of TK-I by purified phosphatase were inhibited by orthovanadate. After dephosphorylation and inactivation by phosphatase, TK-I could be rephosphorylated and reactivated on incubation with ATP. These results suggest that this protein-phosphotyrosine phosphatase may be involved in the regulation of the kinase activity of TK-I.
Swarup
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Centre for Cellular and Molecular Biology, Hyderabad, India.
Subrahmanyam
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D000085:Acetates / Q000494:pharmacology; D019342:Acetic Acid; D000818:Animals; D002413:Cations, Divalent; D002850:Chromatography, Gel; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D036341:Intercellular Signaling Peptides and Proteins; D008970:Molecular Weight; D017830:Octoxynol; D010455:Peptides / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011092:Polyethylene Glycols / Q000494:pharmacology; D017027:Protein Tyrosine Phosphatases; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D012969:Sodium Fluoride / Q000494:pharmacology; D013154:Spleen / Q000201:enzymology*; D014638:Vanadates / Q000494:pharmacology
D000085:Acetates; D002413:Cations, Divalent; D003902:Detergents; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; C027606:L-glutamic acid-L-tyrosine copolymer; D014638:Vanadates; D011092:Polyethylene Glycols; D012969:Sodium Fluoride; D017830:Octoxynol; D011505:Protein-Tyrosine Kinases; D010749:Phosphoprotein Phosphatases; D017027:Protein Tyrosine Phosphatases; D019342:Acetic Acid