5 p-CHOLESTANE · 3 a , 27-DIOL : NAD + OXIDOREDUCTASE AND 3 a-HYDROXY-5 p-CHOLESTAN-27-AL : NAD + OXIDOREDUCT ASE IN RAT LIVER EXTRACT * )
Using rat liver alcohol:NAD+ oxidoreductase and aldehyde:NAD+ oxidoreductase, the following facts were found: 1) 3a-hydroxy-5~-cholestan-27-al was reduced to 5fi -cholestane-3a, 27-diol by liver alcohol: NAD+ oxidoreductase. 2) The optimum pH was pH 7. 0 with potassium phosphate buffer and pH 8. 5 with Tris buffer. 3) The Km value of the substrate to the enzyme was 14. 3 μM. 4) 3a-Hydroxy5fi-cholestan-27al was oxidized to 3a-hydroxy5fi-cholestan27-oic acid by liver aldehyde: NAD+ oxidoreductase. 5) The optimum pH was pH 8. 5 with Tris buffer. 6) The Km value of the substrate to the enzyme was 26. 3 μM.
Horse liver 3 ,7 ,12 -trihydroxy-5 -cholestan-26-al dehydrogenase as a liver aldehyde dehydrogenase.
Abstract 3α,7α,12α-Trihydroxy-5β-cholestan-26-al dehydrogenase (3α,7α,12α-trihydroxy-5β-cholestan-26-al + NAD+ + H2O → 3α,7α,12α-trihydroxy-5β-cholestan-26-oic acid + NADH + H+) in a soluble fraction of horse liver was studied. 1. 1. Horse liver 3α,7α,12α-trihydroxy-5β-cholestan-26-al dehydrogenase consists of two components, the major one of which was purified about 70-fold by (NH4)2SO4 fractionation, gel filtration, ion-exchange column chromatography and hydroxylapatite column chromatography. 2. 2. 3α,7α,12α-trihydroxy-5β-cholestan-26-al dehydrogenase was always accompanied by liver aldehyde:NAD oxidoreductase (EC 1.2.1.3) activity, and the ratio of these two enzyme activities was not altered by purification. 3. 3. Both 3α,7α,12α-trihydroxy-5β-cholestan-26-al dehydrogenase and horse liver aldehyde:NAD+ oxidoreductase were reduced to a similar extent on inactivation of the purified enzyme by heating or by incubation with p-chloromercuribenzoate. 4. 4. The Km value for NAD+ of 3α,7α,12α-trihydroxy-5β-cholestan-26-al dehydrogenase was found to be roughly equal to that of liver aldehyde:NAD+ oxidoreductase. 5. 5. The possibility was discussed that both 3α,7α,12α-trihydroxy-5β-cholestan-26-al dehydrogenase and liver aldehyde:NAD+ oxidoreductase activities may be due to a single protein.
The dehydrogenation of 5β-cholestane-3α, 7α, 12α,26-tetrol by rat liver
Abstract The dehydrogenation of 5β-cholestane-3α, 7α, 12α, 26-tetrol (an intermediate in the conversion of cholesterol to cholic acid) was studied. 1. 1. Experimental conditions for the study of dehydrogenation of 5β-cholestane-3α, 7α, 12α, 26-tetrol were established. 2. 2. When NAD+ was used as an acceptor, the 100 000 × g supernatant fraction of rat liver homogenate showed the highest activity for the dehydrogenation of 5β-cholestane-3α, 7α, 12α, 26-tetrol to 5β-cholestane-3α, 7α, 12α-triol-26-oic acid, and the microsomal fraction revealed a fairly high activity, whereas the mitochondrial fraction did not show any appreciable activity. 3. 3. When 5β-cholestane-3α, 7α, 12α, 26-tetrol was incubated with rat liver 100000 × g supernatant using NAD+ as an acceptor, two moles of NAD+ were reduced to NADH per mole of 5β-cholestane-3α, 7α, 12α-triol-26-oic acid formed from 5β-cholestane-3α, 7α, 12α, 26-tetrol. 4. 4. Among the rat tissues studied only liver showed an appreciable activity for the oxidation of 5β-cholestane-3α, 7α, 12α, 26-tetrol to 5β-cholestane-3α, 7α, 12α-triol-20-oic acid when NAD+ was used as an acceptor.