Studies on the mechanism and regulation of C-4 demethylation in cholesterol biosynthesis. The role of adenosine 3′:5′-cyclic monophosphate

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Abstract
1. An assay for demethylation has been developed based on the release of tritium from 4,4-dimethyl[3α-3H]cholest-7-en-3β-ol (II). 2. The maximum release of 3H from 3α-3H-labelled compound (II) in a rat liver microsomal preparation occurs in the presence of NADPH and NAD+ under aerobic conditions. 3. Incubation of 3α-3H-labelled compound (II) with NADPH under aerobic conditions leads to the formation of a 3α-3H-labelled C-4 carboxylic acid. This compound undergoes dehydrogenation on subsequent anaerobic incubation with NAD+. 4. The 3H released from the steroid was located in [4-3H]nicotinamide and the medium. Incubation with synthetic [4-3H2]NADH gave a similar result. 5. In the presence of glutamate dehydrogenase and α-oxoglutarate part of the 3H released from the steroid was transferred to glutamate. 6. A series of 3-oxo steroids were reduced equally well by [4-3H2]NADH and [4-3H2]NADPH. The reduction of 5α-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 3β-hydroxy dehydrogenase enzyme in the demethylation reaction.

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