Purification of NADPH-dependent dehydroascorbate reductase from rat liver and its identification with 3 alpha-hydroxysteroid dehydrogenase.
Rat liver cytosol has been found to reduce dehydroascorbic acid (DHAA) to ascorbic acid in the presence of NADPH. The enzyme responsible for such activity has been purified by ammonium sulphate fractionation, DEAE-Sepharose, Sephadex G-100 SF and Reactive Red column chromatography, with an overall recovery of 27%. SDS/PAGE of the purified enzyme showed one single protein band with an M(r) of 37,500. A similar value (36,800) was found by gel filtration on a Sephadex G-100 SF column. The results indicate that the enzyme is a homogeneous monomer. The Km for DHAA was 4.6 mM and the Vmax. was 1.55 units/mg of protein; for NADPH Km and Vmax. were 4.3 microM and 1.10 units/mg of protein respectively. The optimum pH was around 6.2. Several typical substrates and inhibitors of the aldo-keto reductase superfamily have been tested. The strong inhibition of DHAA reductase effected by steroidal and non-steroidal anti-inflammatory drugs, together with the ability to reduce 5 alpha-androstane-3,17-dione strongly, suggest the possibility that DHAA reductase corresponds to 3 alpha-hydroxysteroid dehydrogenase. Microsequence analysis performed on the electro-transferred enzyme band shows that the N-terminus is blocked. Internal primary structure data were obtained from CNBr-derived fragments and definitely proved the identity of NADPH-dependent DHAA reductase with 3 alpha-hydroxysteroid dehydrogenase.
Del Bello
B
B
Instituto di Patologia Generale, Università di Siena, Italy.
Maellaro
E
E
Sugherini
L
L
Santucci
A
A
Comporti
M
M
Casini
A F
AF
D015096:3-Hydroxysteroid Dehydrogenases / Q000737:chemistry* / Q000378:metabolism; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D000595:Amino Acid Sequence; D000818:Animals; D002845:Chromatography; D002850:Chromatography, Gel; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology*; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009249:NADP / Q000494:pharmacology*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D013379:Substrate Specificity
D010446:Peptide Fragments; D009249:NADP; D010088:Oxidoreductases; D015096:3-Hydroxysteroid Dehydrogenases; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); C020666:glutathione dehydrogenase (ascorbate); D003488:Cyanogen Bromide