Purification and characterization of salicylhydroxamic acid reductase from rat liver.
Salicylhydroxamic acid reductase, which catalyzes the reduction of salicylhydroxamic acid to salicylamide, was purified from rat liver cytosol. The purification procedure consisted of fractionation with ammonium sulfate, chromatography with Phenyl-Toyopearl 650, DEAE-cellulose, hydroxyapatite, and Sephadex G-200, and chromatofocusing with PBE94. The molecular weight of the enzyme was estimated to be about 140,000 by Sephadex G-200 gel filtration and 152,000 by polyacrylamide gel electrophoresis. The enzyme was dissociated into two different subunits with estimated molecular weights of 41,000 and 32,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These facts suggested that the enzyme is a heterotetramer consisting of two pairs of two nonidentical polypeptide chains. The Km value of the enzyme for salicylhydroxamic acid was estimated to be 91.5 or 88.7 microM in the presence of NADH or NADPH, respectively. The isoelectric point of the enzyme is pH 5.4. The enzyme was highly specific for salicylhydroxamic acid, but it also showed some activity with other hydroxamic acids such as nicotinohydroxamic acid and N-hydroxy-2-acetylaminofluorene. The enzyme activity was inhibited by allopurinol, oxipurinol, dicumarol, menadione, p-chloromercuribenzoic acid, sodium arsenite, potassium cyanide, cupric sulfate, and disulfiram, but little inhibition was observed with oxygen.
Katsura
H
H
Institute of Pharmaceutical Science, Hiroshima University School of Medicine, Japan.
Kitamura
S
S
Tatsumi
K
K
D000818:Animals; D003600:Cytosol / Q000201:enzymology; D004285:Dogs; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012377:Rodentia; D012457:Salicylamides / Q000378:metabolism*; D013045:Species Specificity; D014018:Tissue Distribution
D012457:Salicylamides; D009243:NAD; D009249:NADP; C005703:salicylhydroxamic acid; D010088:Oxidoreductases