Appearance of extracellular glutathione peroxidase (eGPx) in the ascite fluid of casein-elicited rats

Glutathione peroxidase (GPx) activity was detected in the ascite fluid of r ats injected intraperitoneally with 2.5% heat-denatured casein solution. Ac tivity in the ascite fluid increased with time after the injection of casei n, and reached a maximum at 24 h. The active component was concentrated wit h successive 35% ammonium sulfate precipitation and Activated Thiol-Sepharo se 4B column chromatography from the ascite fluid of rats at 24 h after the injection of casein, No N-terminal amino acid of the protein corresponding to GPx was detected by automatic amino acid sequence analysis following se paration with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SD S-PAGE) and transfer to a polyvinyl difluoride (PVDF) membrane, Following B rCN treatment of the protein, the N-terminal amino acid sequences of two 14 and 2.6 kDa peptide fragments were found to be S-G-T-I-Y-E-Y-G-A-L and K-I -H-D-I-R-W-N-F-E, respectively, The former and the latter fragments corresp onded to sequences beginning at the 37th and 176th amino acid residues of r at extracellular GPx (eGPx), respectively. The exclusive presence of eGPx i n the ascite fluid of rats elicited by casein was confirmed immunologically by ELISA, immuno-precipitation and Western blotting assays. No other GPx i sozymes such as cytosolic GPx (cGPx), phospholipid hydroperoxide GPx (PHGPx ) or intestinal GPx (iGPx) were detected, eGPx activity and protein were al so detected in the pleuritic fluid of rats following injection of 2% carrag eenan. These findings indicate that eGPx appears at various sites of acute inflammation in rats. This pattern is due to leakage from circulation as a result of the increased capillary permeability at inflammation sites elicit ed by chemotactic factors.