Redox capacity of cells affects inactivation of glutathione reductase by nitrosative stress.
Glutathione reductase (GR) plays a pivotal role in maintaining glutathione (GSH) in its reduced form. We have isolated a cDNA for rat GR and constructed a baculovirus system to produce recombinant GR on a large scale. This protein was purified by simple, two-step chromatographic procedure using DE52 and 2',5'-ADP Sepharose. Tissue distributions of GR were examined by Northern and Western blotting with a rabbit antibody to purified GR. GR was expressed in the order of reactivity; kidney, colon, liver, stomach, etc. Western blot analysis showed that both the cytosolic and the mitochondrial fractions of liver homogenate gave immunoreactive bands of similar size. This indicates that the same gene products exist in these fractions. Since nitric oxide (NO) produced under inflammatory conditions causes nitrosative stress and affects the redox states of surrounding tissues, we investigated the effects of NO donors on the enzymatic activities of purified GR. S-nitrosoglutathione (GSNO), 3-morpholinosydnonimine N-ethylcarbamide (SIN-1), and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) at 1 mM gave 39, 15, and 12% inhibitions, respectively. In RAW 264.7 cells the GR activity was reported to be inhibited by GSNO. In A549 cells, however, no such change in the activity, protein levels and mRNA of GR was noted. Since these cells have a much higher redox capacity than RAW 264.7 cells as judged by GR activity and thioredoxin reductase activity it wound minimize cellular damage, including inactivation of GR caused by nitrosative stress.
Fujii
T
T
Department of Biochemistry, Osaka University Medical School, Suita, Japan.
Hamaoka
R
R
Fujii
J
J
Taniguchi
N
N
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D005980:Glutathione Reductase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D020030:Nitric Oxide Donors / Q000494:pharmacology; D010084:Oxidation-Reduction; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D014018:Tissue Distribution
D017931:DNA Primers; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D020030:Nitric Oxide Donors; D011994:Recombinant Proteins; D005980:Glutathione Reductase