Characterization of mammalian thioredoxin reductase, thioredoxin and glutaredoxin by immunochemical methods.
Specific polyclonal antibodies towards the oxidized form of bovine thioredoxin reductase (TR) have been obtained in rabbits, and purified. The antigenicity was lost upon reduction of TR by NADPH indicating a large conformational change upon reduction of the redox-active disulfide in the enzyme. The antibodies did not cross-react with other bovine NADPH-dependent dehydrogenases. No reactivity was observed with TR from bacteria, yeast or rat and only a slight reaction was obtained with TR from horse. Immunoaffinity purified anti-thioredoxin and anti-glutaredoxin antibodies were used to develop competitive indirect ELISA assays that were validated giving very good linearity, reproducibility, sensitivity and parallelism. The glutaredoxin (Grx) immunoassay is the first quantitative method described to measure the protein. When applied to a battery of calf tissues the contents of Grx varied from 7 to 120 micrograms per gram of fresh tissue. Skeletal and heart muscles gave the lowest values and spleen and salivary glands the highest. However, skeletal muscle showed the highest gluthathione-hydroxyethyl disulfide oxidoreductase specific activity.
Martínez-Galisteo
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E
Department of Biochemistry and Molecular Biology, Faculty of Veterinary, University of Córdoba, Spain.
Padilla
C A
CA
Holmgren
A
A
Bárcena
J A
JA
D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay / Q000706:statistics & numerical data; D054477:Glutaredoxins; D007150:Immunohistochemistry; D018482:Muscle, Skeletal / Q000737:chemistry; D009206:Myocardium / Q000737:chemistry; D009249:NADP / Q000494:pharmacology; D010088:Oxidoreductases; D011506:Proteins / Q000032:analysis*; D015203:Reproducibility of Results; D012469:Salivary Glands / Q000737:chemistry; D012680:Sensitivity and Specificity; D013154:Spleen / Q000737:chemistry; D013880:Thioredoxin-Disulfide Reductase / Q000032:analysis* / Q000276:immunology; D013879:Thioredoxins / Q000032:analysis*
D000906:Antibodies; C516007:Glrx protein, rat; D054477:Glutaredoxins; D011506:Proteins; D013879:Thioredoxins; D009249:NADP; D010088:Oxidoreductases; D013880:Thioredoxin-Disulfide Reductase