Isolation and characterization of rabbit liver xenobiotic carbonyl reductases

Abstract

Xenobiotic carbonyl reductases have been isolated from rabbit liver by ammonium sulfate fractionation and isoelectric focusing. Although these enzymes are very heterogeneous, the above procedures resolve the majority of the reductases in good yield. Most of the carbonyl reduction of oxisuran, 3,7-dimethyl-1-(5-oxyhexyl)-xanthine, metyrapone and daunorubicin (pH6.0) was accomplished by two distinct enzymes of pI 4.84 and 4.98. Other reductases with lesser activities toward these same substrates also occurred at higher pI values. Also resolved were several forms of enzymes that reduced daunorubicin (pH8.5) (previously identified as aldehyde reductase), naloxone and naltrexone (dihydrornorphinone reductases), and the model compounds, p-nitrobenzaldehyde and p-nitroacetophenone. The hydrogen stereospeciflcity of each of the rabbit liver carbonyl reductases, as well as rat liver aldehyde reductase, was determined by reducing the carbonyl substrates with A- and B-labeled [4- 3H]NADPH and examining transfer of label to alcohol products and retention of label in the resulting oxidized cofactors. All of the oxisuran, metyrapone and daunorubicin (pH 6.0) reductases displayed B-hydrogen stereospecificity. Some enzymes that reduce 3,7-dimethyl-1-(5-oxyhexyl)-xanthine, p-nitroacetophenone and p-nitrobenzaldehyde were also B-stereospecific while other forms of these same enzymes were A-stereospecific. Only daunorubicin (pH8.5) (rabbit and rat), naloxone and naltrexone reductases were exclusively A-stereospecific. Apparent deuterium isotope effects of A- and B-labeled [4- 2H]NADPH with daunorubicin (pH 6.0) reductases, daunorubicin (pH 8.5) reductase and naloxone reductases confirm the above hydrogen stereospecificity assignments. The results confirm the hydrogen specificity of aldehyde reductases as A-stereospecific and the majority of ketone reductases as B-stereospecific. In addition, several significant A-stereospecific ketone reductases appear to represent exceptions to the generalization that enzymes which catalyze the same reaction have the same stereospecificity. Finally, the binding of rat liver aldehyde reductase to NADPH produced a red shift in the cofactor 340 nm absorbance maximum which is opposite to that predicted on the basis of its hydrogen stereospecificity.