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.
The formation of lithocholic acid, chenodeoxycholic acid and other bile acids from 3 beta-hydroxychol-5-enoic acid in vitro and in vivo.
Abstract Previous work has shown that 3β-hydroxycholenoic acid and several naturally occurring C 24 bile acids are formed from cholesterol incubated with rat-liver mitochondria. It is now shown that [ 3 H]3β-hydroxycholenoic acid is converted into lithocholic, chenodeoxycholic, 3α,6β-dihydroxycholanoic and β-muricholic acids in vitro . This suggests that 3β-hydroxycholenoic acid is an intermediate in the formation of C 24 bile acids from cholesterol in vitro . When [ 3 H]3β-hydroxycholenoic acid is injected intravenously into rats, 80% of the radioactivity is recovered in the bile within 2 h, mainly as lithocholic, chenodeoxycholic, 3α,6β-dihydroxycholanoic and β-muricholic acids. This raises the possibility that 3β-hydroxycholenoic acid lies on one of the pathways by which chenodeoxycholic acid is formed in vivo .
Horse liver aldehyde dehydrogenase. I. Purification and characterization.
Abstract Horse liver aldehyde: NAD oxidoreductase (EC 1.2.1.3) has been purified to homogeneity by a procedure consisting of salt fractionation, ion exchange chromatography, and isoelectric focusing. The purified material has a turnover number of 1.85 µmoles of NADH per min per mg of protein when assayed at pH 9.0 with propionaldehyde as substrate. Values obtained for the molecular weight of the native enzyme by sucrose density centrifugation, sedimentation equilibrium, and multiple porosity disc gel electrophoresis were 220,000, 260,000, and 252,000, respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated a subunit molecular weight of 57,000, suggesting a tetrameric structure for the native enzyme. Specificity studies indicated that both aromatic and aliphatic aldehydes were oxidized. For most aldehydes tested, the Michaelis constants were between 0.1 to 1 µm when corrected for equilibrium concentrations of inactive hydrated aldehyde. Chloral, which is completely hydrated, was an inhibitor of the dehydrogenase reaction. Despite the broad aldehyde specificity, substrate analogues in which the aldehydic hydrogen of RCHO was replaced by NH2, CH3, or even OH were not found to be inhibitors.