The major selenium-containing protein in human peripheral granulocytes.
Previously, a selenium-containing protein with subunit molecular weight of 15 kDa was found in peripheral human granulocytes. In continuation of this work, the present communication accounts for purification, identification, and characterization of this major selenium-containing protein. The protein was purified on a heparin-Sepharose column followed by Sephacryl S-200 column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis visualized two bands with subunit molecular weights around 15 kDa. o-Phthaldialdehyde precolumn derivatization and reverse-phase high-performance liquid chromatography showed that the protein contains selenocysteine or selenocystine residues. High-performance gel filtration and isoelectric focusing revealed that the protein had an apparent molecular weight of 32 kDa and a pI value of 7.9. The addition of the protein synthesis inhibitor puromycin to the cell culture medium decreased the 15-kDa protein synthesis. These data suggest that the major selenium-containing protein in peripheral human granulocytes might be a protein with two subunits around 15 kDa. Enzyme studies showed that the protein had peroxidase activity assayed with H2O2 as a substrate and O-dianisidine as a hydrogen donor. This enzymatic activity competed with glutathione peroxidase on the consumption of H2O2, leading to an "inhibiton" of glutathione peroxidase (GSH-Px) activity. Sodium azide could eliminate the inhibition of the protein to GSH-Px. All of the above results implicated that the protein might be a H2O2-dependent selenium containing peroxidase different from GSH-Px. Therefore, the biological function of the protein could be related to eliminating H2O2 generated in the respiratory burst reaction of granulocytes, thus protecting these cells from oxidative damage during phagocytosis.
D001798:Blood Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005979:Glutathione Peroxidase / Q000378:metabolism; D006098:Granulocytes / Q000737:chemistry* / Q000201:enzymology; D006801:Humans; D007525:Isoelectric Focusing; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D011691:Puromycin / Q000494:pharmacology; D051140:Selenoproteins