Dexamethasone induced expression of phosphatase inhibits generation of reactive oxygen species in Ehrlich ascites tumor cells.
Both pre-activated and phorbol ester tetradecanoyl phorbol myristate acetate (TPA) activated reactive oxygen species (ROS) generation were inhibited by dexamethasone in vivo. Time kinetics on influence of dexamethasone on cytosolic phosphoprotein phosphatase activity revealed that, when compared to phosphatase activity in cytosol of control Ehrlich ascites tumor (EAT) cells, a 5-fold increase in specific activity is seen in the cytosol of EAT cells treated (in vivo, 0-90 min. 1 mg/kg body weight) with dexamethasone. Dexamethasone induced phosphatase was partially purified by conventional ion-exchange and gel filtration column chromatographic techniques. Purified phosphatase had a molecular weight of 70 KDa by SDS-PAGE. A dose-dependent inhibition of TPA activated ROS generation by partially purified phosphatase in permeabilized EAT cells suggested that dephosphorylation is a major regulatory mechanism in "switching off" of the respiratory burst. Anti-phosphatase antibodies were raised, purified and were used to quantitate cytosolic phosphatase by ELISA, which revealed that dexamethasone induces 6-fold increase in expression of phosphatase in EAT cells by 120 min. The expression of phosphatase in EAT cell cytosol was further confirmed by immunostaining using anti-phosphatase antibodies, the results of which showed intense blue staining on development with BCIP/NBT.
Salimath
B P
BP
Department of Biochemistry, Manasagangotri, University of Mysore, India.
Sharma
R
R
Tabassum
A
A
D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology; D003907:Dexamethasone / Q000494:pharmacology*; D004790:Enzyme Induction; D004797:Enzyme-Linked Immunosorbent Assay; D007700:Kinetics; D051379:Mice; D010749:Phosphoprotein Phosphatases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D016897:Respiratory Burst; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D017382:Reactive Oxygen Species; D003907:Dexamethasone; D010749:Phosphoprotein Phosphatases; D013755:Tetradecanoylphorbol Acetate