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 rats injected intraperitoneally with 2.5% heat-denatured casein solution. Activity in the ascite fluid increased with time after the injection of casein, and reached a maximum at 24 h. The active component was concentrated with successive 35% ammonium sulfate precipitation and Activated Thiol-Sepharose 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 separation with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transfer to a polyvinyl difluoride (PVDF) membrane. Following BrCN 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 corresponded to sequences beginning at the 37th and 176th amino acid residues of rat extracellular GPx (eGPx), respectively. The exclusive presence of eGPx in the ascite fluid of rats elicited by casein was confirmed immunologically by ELISA, immuno-precipitation and Western blotting assays. No other GPx isozymes such as cytosolic GPx (cGPx), phospholipid hydroperoxide GPx (PHGPx) or intestinal GPx (iGPx) were detected. eGPx activity and protein were also detected in the pleuritic fluid of rats following injection of 2% carrageenan. 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 elicited by chemotactic factors.