Shigeoka S, Takeda T, Hanaoka T
Department of Food and Nutrition, Kinki University, Nara, Japan.
Biochem J. 1991 May 1;275 ( Pt 3)(Pt 3):623-7. doi: 10.1042/bj2750623.
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.
通过聚丙烯酰胺凝胶电泳（PAGE）判断，莱茵衣藻中由亚硒酸盐诱导产生的谷胱甘肽过氧化物酶已被纯化了约323倍，产率为10%。天然酶的相对分子质量为67,000，由四个相对分子质量为17,000的相同亚基组成。谷胱甘肽是唯一的电子供体，每毫克蛋白质的比活性为193.6 μmol/分钟。L-抗坏血酸、NADH、NADPH、邻苯三酚、愈创木酚和邻联茴香胺均不能向该酶提供电子。除了过氧化氢外，该酶还能还原有机氢过氧化物。谷胱甘肽和过氧化氢的米氏常数分别为3.7 mM和0.24 mM。酶反应按乒乓双底物机制进行。氰化物和叠氮化物对活性无影响。该酶每摩尔蛋白质约含3.5个硒原子。免疫沉淀时，莱茵衣藻谷胱甘肽过氧化物酶会沉淀，针对牛红细胞谷胱甘肽过氧化物酶产生的抗体可使其活性受到约90%的抑制。在蛋白质印迹SDS/PAGE中，该抗体也能与莱茵衣藻谷胱甘肽过氧化物酶的亚基发生交叉反应。就酶学、物理化学和免疫学性质而言，实验结果清楚地表明，莱茵衣藻谷胱甘肽过氧化物酶与其他已充分表征的含硒动物源谷胱甘肽过氧化物酶相似。
