Purification and characterization of glutathione-dependent dehydroascorbate reductase from rat liver.
GSH-dependent enzymic reduction of dehydroascorbic acid to ascorbic acid has been studied in rat liver cytosol. After gel filtration of cytosol on Sephadex G-100 SF, dehydroascorbate reductase activity was recovered in two distinct peaks, one corresponding to glutaredoxin (an enzyme already known for its dehydroascorbate reductase activity) and another, much larger one, corresponding to a novel enzyme different from glutaredoxin. The latter was purified to apparent homogeneity. The purification process involved (NH4)2SO4 fractionation, followed by DEAE-Sepharose, Sephadex G-100 SF and Reactive Red chromatography. SDS/PAGE of the purified enzyme in either the presence or absence of 2-mercaptoethanol demonstrated a single protein band of M(r) 31,000. The M(r) determined by both Sephadex G-100 SF chromatography and h.p.l.c. was found to be approx. 48,000. H.p.l.c. of the denatured enzyme gave an M(r) value identical with that obtained by SDS/PAGE (31,000). The apparent Km for dehydroascorbate was 245 microM and the Vmax. was 1.9 mumol/min per mg of protein; for GSH they were 2.8 mM and 4.5 mumol/min per mg of protein respectively. The optimal pH range was 7.5-8.0. Microsequence analysis of the electro-transferred enzyme band showed that the N-terminus is blocked. Data on internal primary structure were obtained from CNBr-and N-chlorosuccinimide-derived fragments. No significative sequence similarity was found to any of the protein sequences contained in the Protein Identification Resource database.
Maellaro
E
E
Instituto di Patologia Generale, Università di Siena, Italy.
Del Bello
B
B
Sugherini
L
L
Santucci
A
A
Comporti
M
M
Casini
A F
AF
D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D003600:Cytosol / Q000201:enzymology; D003683:Dehydroascorbic Acid / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005978:Glutathione / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology*; D008297:Male; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D017421:Sequence Analysis; D013388:Succinimides
D010446:Peptide Fragments; D013388:Succinimides; C015752:N-chlorosuccinimide; D010088:Oxidoreductases; C020666:glutathione dehydrogenase (ascorbate); D005978:Glutathione; D003488:Cyanogen Bromide; D003683:Dehydroascorbic Acid