Formation and isolation of delta 5-3-ketosteroids using a purified rat liver alcohol dehydrogenase.
3 beta-Hydroxy-5-androsten-17-one is converted to 5-androstene-3, 17-dione by rat liver alcohol dehydrogenase (ADH). We have reported on the purity of the enzyme which is eluted with pyrazole as a single homogeneous protein using an AMP-agarose affinity column. Rat liver ADH can oxidize hydroxyl groups not only at 3 beta-, but also at 3 alpha-, and 17 beta-positions to a lesser extent; thus it is a pure mammalian enzyme with multifunctional activity for steroids. Since it does not contain delta 5-isomerase activity, the reaction of the dehydrogenase to form the delta 5-ketosteroid intermediate can be observed at pH 7.0, 25 degrees C. Similarly, intermediary product, 5-pregnene-3,20-dione, can be isolated in the conversion of pregnenolone by ADH to progesterone. With buffer alone in a cuvette, a non-enzymatic isomerization of the delta 5-3-ketone occurs at a slow rate (t 1/2 = 6 hrs) but occurs rapidly during isolation procedures. The delta 5-3-ketosteroid intermediates were identified by their behavior on TLC plates with UV light and by their characteristic spectra in the NMR.
D000426:Alcohol Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D003687:Dehydroepiandrosterone / Q000378:metabolism; D007664:Ketosteroids / Q000096:biosynthesis* / Q000302:isolation & purification; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D010084:Oxidation-Reduction; D011284:Pregnenolone / Q000378:metabolism; D051381:Rats
D007664:Ketosteroids; D003687:Dehydroepiandrosterone; D011284:Pregnenolone; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase