Purification of NADPH-linked alpha,beta-ketoalkene double bond reductase from rat liver.
NADPH-linked alpha,beta-ketoalkene double bond reductase was purified from rat liver cytosol by fractionation with ammonium sulfate, and chromatography with DEAE-cellulose. AF-Blue Toyopearl and hydroxyapatite. The purified enzyme was homogeneous by the criterion of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 39,500 by the electrophoresis and by HPLC gel filtration on a TSK gel G3000 SWXL column. The double bond of 2-alkenals was also reduced by the enzyme, but to a lesser extent. The enzyme activity was inhibited by 5,5'-dithiobis(2-nitrobenzoic acid), p-chloromercuribenzoic acid, N-ethylmaleimide, iodoacetamide, dicumarol, quercitrin, and disulfirum. However, the enzyme was insensitive to oxygen.
Kitamura
S
S
Institute of Pharmaceutical Science, Hiroshima University School of Medicine, Japan.
Tatsumi
K
K
D000818:Animals; D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003600:Cytosol / Q000201:enzymology; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D006168:Guinea Pigs; D006882:Hydroxyapatites; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008647:Mesocricetus; D051379:Mice; D008815:Mice, Inbred Strains; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D013045:Species Specificity; D013379:Substrate Specificity
D006882:Hydroxyapatites; D009249:NADP; D017886:Durapatite; D010088:Oxidoreductases; C040723:alpha,beta-ketoalkene reductase