Characterization and immunological properties of selenium-containing glutathione peroxidase induced by selenite in Chlamydomonas reinhardtii.
The selenite-induced glutathione peroxidase in Chlamydomonas reinhardtii has been purified about 323-fold with a 10% yield, as judged by PAGE. The native enzyme had an Mr of 67,000 and was composed of four identical subunits of Mr 17,000. Glutathione was the only electron donor, giving a specific activity of 193.6 mumol/min per mg of protein. L-Ascorbate, NADH, NADPH, pyrogallol, guaiacol and o-dianisidine did not donate electrons to the enzyme. In addition to H2O2, organic hydroperoxides were reduced by the enzyme. The Km values for glutathione and H2O2 were 3.7 mM and 0.24 mM respectively. The enzyme reaction proceeded by a Ping Pong Bi Bi mechanism. Cyanide and azide had no effect on the activity. The enzyme contained approx. 3.5 atoms of selenium per mol of protein. On immunoprecipitation, Chlamydomonas glutathione peroxidase was precipitated and its activity was inhibited about 90% by the antibody raised against bovine erythrocyte glutathione peroxidase. The antibody also cross-reacted with the subunits of Chlamydomonas glutathione peroxidase in Western blotting SDS/PAGE. In terms of enzymic, physico-chemical and immunological properties, the experimental results demonstrate clearly that Chlamydomonas glutathione peroxidase resembles other well-characterized glutathione peroxidases from animal sources that contain selenium.
Shigeoka
S
S
Department of Food and Nutrition, Kinki University, Nara, Japan.
Takeda
T
T
Hanaoka
T
T
D000818:Animals; D000906:Antibodies / Q000276:immunology; D000941:Antigens / Q000276:immunology; D015153:Blotting, Western; D002417:Cattle; D002696:Chlamydomonas / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction / Q000187:drug effects; D004912:Erythrocytes / Q000201:enzymology; D005978:Glutathione / Q000378:metabolism; D005979:Glutathione Peroxidase / Q000032:analysis / Q000096:biosynthesis* / Q000276:immunology; D006861:Hydrogen Peroxide / Q000378:metabolism; D007163:Immunosorbent Techniques; D008970:Molecular Weight; D020887:Selenious Acid; D012643:Selenium / Q000032:analysis* / Q000494:pharmacology*; D013379:Substrate Specificity
D000906:Antibodies; D000941:Antigens; D006861:Hydrogen Peroxide; D005979:Glutathione Peroxidase; D020887:Selenious Acid; D005978:Glutathione; D012643:Selenium