Purification and properties of bovine thioredoxin system.
Using a variety of chromatographic techniques, a crude extract from bovine liver was fractionated to obtain pure preparations of thioredoxin reductase, thioredoxin, glutaredoxin and glutathione reductase with good yields. The turbidimetric assay of thioredoxin with insulin as the disulfide substrate was optimized; by incorporation of the lag time (tau) into the calculations, linearity was maintained for a wider range of thioredoxin concentrations, and a distinction could be made between reduced and non-reduced forms. Subunit composition and molecular mass, absorption spectrum and kinetic parameters of thioredoxin reductase were similar to those of other mammalian thioredoxin reductases. By chromatofocusing, two peaks of activity were detected at pH 5.5 and 5.8. Structural changes undergone by the thioredoxin molecule upon oxido-reduction were detected by isoelectric focusing, with a shift of 0.1 pH unit of its pI, and by analytical anion exchange chromatography, with a conspicuous shift of its retention time. These two methods also revealed the presence of a form of thioredoxin not undergoing the above mentioned redox-mediated structural shifts that accounted for > 75% of the total activity.
Martínez-Galisteo
E
E
Departamento de Bioquímica y Biología Molecular, Facultad de Veterinaria, Universidad de Córdoba, Spain.
Padilla
C A
CA
García-Alfonso
C
C
López-Barea
J
J
Bárcena
J A
JA
D000818:Animals; D002417:Cattle; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D054477:Glutaredoxins; D005980:Glutathione Reductase / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology / Q000378:metabolism*; D009391:Nephelometry and Turbidimetry; D010084:Oxidation-Reduction; D010088:Oxidoreductases; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D013880:Thioredoxin-Disulfide Reductase / Q000302:isolation & purification / Q000378:metabolism*; D013879:Thioredoxins / Q000302:isolation & purification / Q000378:metabolism*
D054477:Glutaredoxins; D011506:Proteins; D013879:Thioredoxins; D010088:Oxidoreductases; D005980:Glutathione Reductase; D013880:Thioredoxin-Disulfide Reductase