Purification and properties of a metyrapone-reducing enzyme from mouse liver microsomes--this ketone is reduced by an aldehyde reductase.
A ketone reducing enzyme was purified to homogeneity from female mouse liver microsomes, using the diagnostic cytochrome P-450 inhibitor metyrapone as a substrate. In contrast to the usually employed indirect spectrophotometric recording of pyridine nucleotide oxidation at 340 nm, a HPLC method was applied for direct alcohol metabolite determination. Purification of the carbonyl reductase resulted in a 360-fold increase in specific activity together with a single band in the 34 kD region after SDS-polyacrylamide gel electrophoresis. Phenobarbital, indomethacin, dicoumarol and 5 alpha-dihydrotestosterone inhibited the enzyme, whereas quercitrin did not affect the enzyme activity. Thus, by inhibitor classification of carbonyl reductases the ketone metyrapone is reduced by an aldehyde reductase, rather than by a ketone reductase. Dihydrotestosterone, the strongest inhibitor, is supposed to be the physiological substrate for the purified enzyme. It was demonstrated that during the steps of purification both NADPH and NADH can supply the required reducing equivalents, although the activity with NADH is weaker. The highest activity was obtained using an NADPH-regenerating system. Ethanol and the nonionic detergent Emulgen 913 led to an increased specific activity, indicating that the enzyme is bound to the membranes of the endoplasmic reticulum in a latent state. From these results it is concluded that the microsomal metyrapone-reducing enzyme belongs to the family of carbonyl reductases, but differs from the common patterns of their classification with regard to cofactor requirement and inhibitor susceptibility.
Maser
E
E
Department of Pharmacology and Toxicology, School of Medicine, University of Marburg, Lahnberge, Federal Republic of Germany.
Netter
K J
KJ
D000426:Alcohol Dehydrogenase / Q000145:classification; D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000145:classification / Q000302:isolation & purification*; D000449:Aldehyde Reductase; D000074408:Aldo-Keto Reductases; D000818:Animals; D005260:Female; D008797:Metyrapone / Q000378:metabolism*; D051379:Mice; D008862:Microsomes, Liver / Q000201:enzymology*; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction
D009249:NADP; D000429:Alcohol Oxidoreductases; D000074408:Aldo-Keto Reductases; D000426:Alcohol Dehydrogenase; D000449:Aldehyde Reductase; C000614209:AKR7A5 protein, mouse; D008797:Metyrapone