J. Biol. Chem., Vol. 265, Issue 1, 108-113, Jan, 1990
Elevation of rat liver mRNA for selenium-dependent glutathione peroxidase by selenium deficiency
NQ Li, PS Reddy, K Thyagaraju, AP Reddy, BL Hsu, RW Scholz, CP Tu and CC Reddy
Department of Veterinary Science, Pennsylvania State University, University Park 16802.
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.
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