Purification and characterisation of chicken liver monomeric glutathione peroxidase.
1. A novel glutathione peroxidase, which is distinct from tetrameric glutathione peroxidase, was purified to homogeneity from a broiler chick liver cytosolic fraction using 5 different column chromatographic methods. 2. The enzyme in cytosol was separated from 'classic' tetrameric glutathione peroxidase and glutathione S-transferases by DEAE-Sephacel and Sephadex G-100 chromatographies and further purified by Mono Q hydroxylapatite and sulphobromophthalein-S-glutathione-agarose chromatographies. 3. The molecular weight of the purified enzyme determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis was 19,500 and that found by gel filtration chromatography was comparable. This indicates that the enzyme protein is a single polypeptide. The isoelectric point of the enzyme was determined as 7.0 by polyacrylamide gel isoelectric focusing. 4. The purified enzyme catalysed the reduction of hydrogen peroxide, cumene hydroperoxide, text-butyl hydroperoxide and linoleic acid hydroperoxide. Furthermore, it reduced phosphatidylcholine hydroperoxide in the absence of phospholipase A2. The optimum pH for the enzyme reaction was 7.0. The antiserum against the purified enzyme reacted with the 19.5 kDa polypeptide in the liver cytosol of duck and quail.
Miyazaki
S
S
Toxico-pharmacology Laboratory, National Institute of Animal Health 3-1-1, Ibaraki, Japan.
Motoi
Y
Y
D000818:Animals; D002645:Chickens; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004372:Ducks; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D005979:Glutathione Peroxidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008297:Male; D008970:Molecular Weight; D011784:Quail; D013379:Substrate Specificity
D007527:Isoenzymes; D046911:Macromolecular Substances; D017886:Durapatite; D005979:Glutathione Peroxidase