New strategies for the isolation and activity determination of naturally occurring type-4 glutathione peroxidase.
Type 4 glutathione peroxidase (GPx4) is a widely expressed mammalian selenoenzyme known to play a vital role in cytoprotection against lipid hydroperoxide (LOOH)-mediated oxidative stress and regulation of oxidative signaling cascades. Since prokaryotes are not equipped to express mammalian selenoproteins, preparation of recombinant GPx4 via commonly used bacterial transformation is not feasible. A published procedure for isolating the enzyme from rat testis employs affinity chromatography on bromosulfophthalein-glutathione-linked agarose as the penultimate step in purification. Since this resin is no longer commercially available and preparing it in satisfactory operational form is tedious, we have developed an alternative purification approach based on sequential anion exchange, size exclusion, and cation exchange chromatography. Final preparations were found to be essentially homogeneous in GPx4 (M(r) approximately 20 kDa), as demonstrated by SDS-PAGE with protein staining and immunoblotting. Specific enzymatic activity was determined using a novel thin-layer chromatographic approach in which the kinetics of phosphatidylcholine hydroperoxide loss or cholesterol-7alpha-hydroperoxide loss was monitored. A >400-fold purification of active enzyme has been attained. The relatively straightforward isolation procedure described should prove valuable for further functional studies on GPx4, e.g. how its ability to catalyze LOOH reduction compares with that of other LOOH detoxifying enzymes.
Kernstock
Robert M
RM
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226-3548, USA.
Girotti
Albert W
AW
D000818:Animals; D001671:Biochemistry / Q000379:methods*; D002384:Catalysis; D002845:Chromatography; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D005979:Glutathione Peroxidase / Q000302:isolation & purification* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008054:Lipid Peroxides / Q000378:metabolism; D008297:Male; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D051381:Rats; D013737:Testis / Q000201:enzymology
D008054:Lipid Peroxides; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D005979:Glutathione Peroxidase