Characterization and immunological properties of selenium-containing glutathione peroxidase induced by selenite in Chlamydomonas reinhardtii

Abstract

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.

A Selenoprotein in the Plant Kingdom

“…Thus, the results presented in this study indicate that CrGPX is different from the one characterized by Shigeoka et al (22). We have found a protein of a different subunit M r , the corresponding gene sequence does not confirm a tetrameric structure, and our GPX did not cross-react with bovine erythrocyte GPX antibodies, kindly provided to us by Dr. M. Lecomte (data not shown).…”

“…Two other reports showed that, in Euglena gracilis, a purified selenium-independent GPX-like protein resembled classical GPX in its activity and structure (46) and, in C. reinhardtii, a selenium-dependent GPX was found to be a tetramer composed of identical subunits (22).…”

“…In all these proteins, only the association of a selenium atom to a protein was determined. In C. reinhardtii, selenium was determined fluorimetrically using 2,3-diaminonaphtalene (22). In A. vera, selenium was determined by atomic absorption spectrometry (24).…”

“…Purified proteins exhibiting selenium-dependent classical GPX activity have been described in Chlamydomonas reinhardtii (21,22), in germinating barley (23), and in Aloe vera (24), but, in all these systems, it has only been shown that an inorganic selenium atom is associated with proteins catalyzing peroxidase activity, and it is well known that selenium can be associated with such molecules in various chemical forms (25). No direct proof has been provided either for the existence of a UGA codon in the genes or for the incorporation of selenium into the amino acid selenocysteine in the corresponding proteins.…”

The Peroxiredoxin and Glutathione Peroxidase Families in Chlamydomonas reinhardtii

“…Early studies had indicated that addition of sodium selenite to Chlamydomonas cultures led to a strong increase in GPX activity in total soluble extracts (Yokota et al 1988). Purification of the corresponding protein revealed that the enzyme was associated with selenium and exhibited biochemical properties comparable to mammalian selenoGPXs (Shigeoka et al 1991). Although these studies suggested the existence of selenoGPX in Chlamydomonas, no DNA or protein sequence was determined.…”