Rat Liver Daunorubicin Reductase: AN ALDO-KETO REDUCTASE

Abstract

Abstract Daunorubicin, a cancer chemotherapeutic antibiotic, is converted by rat liver extracts to daunorubicinol by daunorubicin reductase. This enzyme is purified from rat liver by ammonium sulfate fractionation, DEAE-cellulose, hydroxyapatite, and Bio-Gel P-200 column chromatography with an overall recovery of 46%. On the basis of polyacrylamide electrophoresis with and without sodium dodecyl sulfate, gel filtration chromatography, and high speed ultracentrifugation, the final preparation is judged homogeneous. Sedimentation equilibrium analysis and sodium dodecyl sulfate polyacrylamide electrophoresis give molecular weights of 38,796 ± 369 and 39,800, respectively, and suggest a single polypeptide chain. In agreement is the molecular weight of 39,541 calculated from the proposed amino acid composition. The enzyme has an isoelectric point of 6.3 and a strict requirement for NADPH. The daunorubicin-NADPH interaction occurs with a 1:1 stoichiometry. The apparent equilibrium constant at pH 8.5 for reduction is 2.91 (± 0.26) x 109 m-1, and the apparent Km for daunorubicin is 57 µm. Several common sulfhydryl reactants inhibited the enzyme. A closely related antibiotic, adriamycin, is reduced at about 5% the rate of daunorubicin reduction. In addition to the antibiotics, the enzyme reduces some aldo sugars, particularly, d-glucuronolactone, d-glyceraldehyde, and the isomers d-glucuronate and d-galacturonate. d-Glucuronolactone and daunorubicin were the preferred substrates. This reactivity with d-glucuronolactone suggests a normal role of daunorubicin reductase in ascorbic acid synthesis or in the glucuronic acid cycle, or both.