Mechanism of C-5 Double Bond Introduction in the Biosynthesis of Cholesterol by Rat Liver Microsomes EVIDENCE FOR THE PARTICIPATION OF MICROSOMAL CYTOCHROME b,

The dehydrogenation reaction of cholest-7-en-3P-ol (I) to cholesta-5,7-diem3/3-ol (II) in the presence of NADH was studied in rat liver microsomes and in microsomal acetone powder preparations, using [3Lu-“Hlcholest-‘7-en-3fi-o1. It was found that the reaction was inhibited by menadione, adenosine diphosphate, potassium ferricyanide, and cytochrome c while p-cresol had no effect. These results indicated the participation of a microsomal electron transport system in the dehydrogenation of cholest-7-en-3P-01. The conversion of cholest-7-en-3P-ol to cholesta-5,7-dien-3p-ol was also observed in the absence of NADH when ascorbic acid was included in the incubation mixture. However, the ascorbic acid-catalyzed dehydrogenation was not inhibited by potassium ferricyanide. Immunological evidence that microsomal cytochrome b, is involved in the dehydrogenation of (I) to (II) was obtained. Antibodies specific for rat liver microsomal cytochrome b, were elicited in rabbits. The anticytochrome 6, immunoglobulin fraction inhibited rat liver microsomal NADH-cytochrome c reductase but not NADPH-cytochrome c reductase. Also, the extent of reduction of cytochrome bj was not affected by the antibodies. The conversion of (I) to (II) by rat liver microsomes was inhibited (73%) by anticytochrome b, immunoglobulin at a ratio of microsomal protein:immunoglobulin of 1:5.6. These results are consistent with the participation of microsomal cytochrome b, in the introduction of the C-5 double bond in cholesterol biosynthesis. A close analogy of the microsomal dehydrogenation of fatty acids and of cholest-7en-3jSol is apparent and this suggests a possible similarity in the mechanisms of the two reactions.