Purification, characterization, and immunolocalization of a thioredoxin reductase from adult Fasciola hepatica.
Thioredoxin reductase (TrxR), an enzyme belonging to the flavoprotein family of pyridine nucleotide-disulfide oxidoreductases, was isolated from the deoxycholate-soluble extract of the common liver fluke, Fasciola hepatica. Purification to homogeneity of the 60-kDa enzyme from the adult worm was achieved by a combination of ammonium sulfate fractionation, anion exchange, and affinity chromatography on 2',5'-adenosine diphosphate-Sepharose. Using the 5,5'-dithiobis(2-nitrobenzoic acid) assay, the purified TrxR showed a specific activity of 7,117 U min(-1) mg(-1). The enzyme activity was completely inhibited by the presence of the gold compound aurothioglucose (IC50 = 120 nm), indicating that F. hepatica TrxR is a selenoenzyme. Also, the enzyme was capable of reducing disulfide bonds in insulin and was activated by the presence of the reduced form of flavin adenine dinucleotide, properties shared with mammalian TrxRs. Furthermore, the isolated enzyme showed very low glutaredoxin (Grx) activity (0.47 U mg(-1)), but no glutathione reductase activity was detected. Affinity-purified IgGs (20 microg ml(-1)) from the antisera produced against the purified TrxR inhibited its activity about 80% with respect to the control. The enzyme was immunolocalized in cells located within the parenchyma and in the testes, but it was not found in the tegument of the adult fluke.
Maggioli
Gabriela
G
Unidad de Biología Parasitaria, Facultad de Ciencias, Instituto de Higiene, Av. A. Navarro 3051, CP 11600 Montevideo, Uruguay.
Piacenza
Lucía
L
Carambula
Beatriz
B
Carmona
Carlos
C
D000818:Animals; D006051:Aurothioglucose / Q000494:pharmacology; D002417:Cattle; D005591:Chemical Fractionation; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005210:Fasciola hepatica / Q000201:enzymology*; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D007074:Immunoglobulin G / Q000276:immunology; D013880:Thioredoxin-Disulfide Reductase / Q000037:antagonists & inhibitors / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism
D004791:Enzyme Inhibitors; D007074:Immunoglobulin G; D006051:Aurothioglucose; D013880:Thioredoxin-Disulfide Reductase