title: Studies on the mechanism and regulation of C-4 demethylation in cholesterol biosynthesis. The role of adenosine 3':5'-cyclic monophosphate.
abstract: 1. An assay for demethylation has been developed based on the release of tritium from 4,4-dimethyl[3alpha-(3)H]cholest-7-en-3beta-ol (II). 2. The maximum release of (3)H from 3alpha-(3)H-labelled compound (II) in a rat liver microsomal preparation occurs in the presence of NADPH and NAD(+) under aerobic conditions. 3. Incubation of 3alpha-(3)H-labelled compound (II) with NADPH under aerobic conditions leads to the formation of a 3alpha-(3)H-labelled C-4 carboxylic acid. This compound undergoes dehydrogenation on subsequent anaerobic incubation with NAD(+). 4. The (3)H released from the steroid was located in [4-(3)H]nicotinamide and the medium. Incubation with synthetic [4-(3)H(2)]NADH gave a similar result. 5. In the presence of glutamate dehydrogenase and alpha-oxoglutarate part of the (3)H released from the steroid was transferred to glutamate. 6. A series of 3-oxo steroids were reduced equally well by [4-(3)H(2)]NADH and [4-(3)H(2)]NADPH. The reduction of 5alpha-cholest-7-en-3-one was shown to use the 4B H atom from the nucleotide. 7. 3':5'-Cyclic AMP was shown to be a competitive inhibitor of the 3beta-hydroxy dehydrogenase enzyme in the demethylation reaction.
mesh_terms: D000332:Aerobiosis; D000693:Anaerobiosis; D000818:Animals; D002264:Carboxylic Acids / Q000096:biosynthesis; D002776:Cholestanes / Q000378:metabolism; D002784:Cholesterol / Q000096:biosynthesis*; D000242:Cyclic AMP / Q000378:metabolism*; D005969:Glutamate Dehydrogenase; D005971:Glutamates; D005977:Glutarates; D006913:Hydroxysteroid Dehydrogenases / Q000037:antagonists & inhibitors; D066298:In Vitro Techniques; D007664:Ketosteroids; D008722:Methods; D008745:Methylation; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009536:Niacinamide; D051381:Rats; D013261:Sterols / Q000378:metabolism; D014316:Tritium
chemical_list: D002264:Carboxylic Acids; D002776:Cholestanes; D005971:Glutamates; D005977:Glutarates; D007664:Ketosteroids; D013261:Sterols; D009243:NAD; D014316:Tritium; D009536:Niacinamide; D009249:NADP; D002784:Cholesterol; D000242:Cyclic AMP; D006913:Hydroxysteroid Dehydrogenases; D005969:Glutamate Dehydrogenase