Abstract
Selenium-dependent glutathione peroxidase (Se-GSH-Px, GSH-H2O2 oxidoreductase EC 1.11.1.9) is the best characterized selenoprotein in higher animals, but the mechanism whereby selenium becomes incorporated into the enzyme protein remains under investigation. To elucidate the mechanism of insertion of selenium into Ge-GSH-Px further, we have systematically analyzed and compared the results of Western blot, in vitro translation immunoprecipitation, and Northern blot experiments conducted with liver proteins and RNAs obtained from rats fed on selenium-deficient and selenium-supplemented diets. The anti-serum employed in this study was raised against an electrophoretically pure Se-GSH-Px preparation obtained from rat livers by a simplified purification procedure involving separation by high performance liquid chromatography on a hydrophobic interaction column. Different forms of Se-GSH-Px, including apo-protein, cross-reacted with this antiserum and Western blot analysis found no Se-GSH-Px protein present in livers from rats fed on selenium-deficient diets. By contrast, a distinct protein band corresponding to purified Se-GSH-Px was detected in livers from selenium-supplemented animals, a result consistent with the finding that the Se-GSH-Px activity was reduced to undetectable levels in livers of selenium-deficient rats. The in vitro translation experiments, however, indicated not only that mRNA for Se-GSH-Px was present during selenium deficiency but also that its translation products contained 2-3-fold as much immunoprecipitable protein as the products of poly(A) RNA from livers of selenium-supplemented rats. This result suggests that the Se-GSH-Px mRNA may be increased in the selenium-deficient state. Elevated levels of Se-GSH-Px mRNA were directly demonstrated in Northern blot experiments employing cDNA clone pGPX1211 as a probe. A similar increase in Se-GSH-Px mRNA was observed in such other tissues as kidney, testis, brain, and lung tissue, in selenium-deficient states. The present data support the co-translational mechanism for the incorporation of selenium into Se-GSH-Px in rat liver.
Highlights
EC 1.11.1.9) is the best characterized selenoprotein in higher animals, but the mechanism whereby selenium becomes incorporated into the enzyme protein remains under investigation
In the present investigation we report evidence for the co-translational insertion of selenium into rat liver Se-GSH-Px, and for the elevation of rat liver mRNA for Se-GSH-Px in a selenium-deficient state
Px in Rut Liuer-The interrelatedness of the metabolic functions of selenium and vitamin E having been established [20, 21], we investigated the effects of altered selenium and vitamin E nutrition on the expression of Se-GSH-Px in rat livers
Summary
Our understanding of the molecular mechanisms underlying the biological activity of selenium is still somewhat limited, but a notable advance occurred in 1973 when selenium was found to be an essential component of mammalian glutathione peroxidase (GSH-Px, GSH:H202 oxidoreductase, EC 1.11.1.9)’ [1, 2]. One of the first enzymes shown to contain selenium, GSHPx is the best characterized selenoprotein in higher animals. This enzyme, which catalyzes the reaction shown in equation I (where R = H or alkyl; GSH = glutathione), plays an important role in eliminating potentially harmful. All of the enzymes known to contain selenocysteine catalyze oxidationreduction reactions, which suggests that oxidized forms of selenium are most likely involved in their catalytic function. A specification of the mechanism by which selenium is incorporated into these proteins remains elusive It is not entirely clear whether this occurs by post-translational [12] or co-translational [13] processes; the recent discovery of an (in-frame) UGA opal nonsense codon at position 47,
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