No abstract

It was shown that 100mug quantities of 4,4'-dimethyl[2-(3)H(2)]cholesta-8,14-dien-3beta-ol (IIIa), tritiated cholesta-8,14-dien-3beta-ol, 4,4'-dimethyl[2-(3)H(2)]cholesta-7,14-dien-3beta-ol, dihydro[2-(3)H(2)]lanosterol and [24-(3)H]lanosterol were converted by a 10000g supernatant of rat liver homogenate into cholesterol in 17%, 54%, 6%, 9.5% and 24% yields respectively. From an incubation of dihydro[3alpha-(3)H]lanosterol with a rat liver homogenate in the presence of a trap up to 38% of the radioactivity was found to be associated with a fraction that was unambiguously shown to be 4,4'-dimethylcholesta-8,14-dien-3beta-ol. Another related compound, 4,4'-dimethylcholesta-7,14-dien-3beta-ol was also shown to be equally effective in its ability to trap compound (IIIa) from an incubation of dihydro[3alpha-(3)H]lanosterol. The mechanism of the further conversion of the compound (IIIa) into cholesterol occurred by the reduction of the 14,15-double bond and involved the addition of a hydrogen atom from the medium to C-15 and another from the 4-position of NADPH to C-14. Two possible mechanisms for the removal of the 14alpha-methyl group in sterol biosynthesis are discussed.

No abstract

It is shown that during the saturation of steroid carbon-carbon double bonds at Delta(24,25) and Delta(14,15) the ;hydride ion' originates from the 4B side of the NADPH.

The conversion of desmosterol into cholesterol was carried out in the presence of either tritiated water or [4-3H2]NADPH. The side chain fragment consisting of carbon atoms 23-27 of cholesterol was obtained through the combination of biological and chemical techniques. Selective degradation of the fragment led to the conclusion that in the saturation of the 24,25-double bond of desmosterol a hydrogen atom from the medium is added to C-24 and another from the 4-position of NADPH to C-25. These results in conjunction with similar studies previously reported on the saturation of other C=C in steroid biosynthesis are interpreted in terms of a general mechanism. It is suggested that the first crucial event in the pyridine nucleotide linked reduction is the activation of the substrate through protonation to give an electron deficient species which in the next step is neutralized by the addition of a hydride from NAD(P)H t o furnish the product.Pyridine nucleotides have been extensively implicated in the reduction of groups such as C=O, C=N and C=C and many pyridine nucleotide linked enzymes have been purified and subjected to extensive kinetic analysis. Much valuable data has been obtained concerning the order of binding of substrates (for review see 111) but these studies have yielded little information concerning the actual mechanism and sequence of bond formation.We envisaged that the orientation of addition of a proton from the medium and a hydride ion from NADPH to olefinic linkages may give information concerning the precise mechanism of C =C reduction reactions. These mechanistic conclusions should have some bearing on the mechanism of substrate activation in other pyridine nucleotide linked reductions involving C=O and C=N.I n this paper we study the reduction of the 24-25 double bond during the conversion of cholesta-5, 24-&en-3p-01 (I) (desmost,erol) into cholesterol (11) and discuss the results obtained from this study and other work we have reported on the reduction of sterol double bonds a t 7-8 and 14-15 The incubation of cholesterol with Tetrahymena pyriformis and the isolation of (111) have been described previously [13]. The yield of (111) from cholesterol in various experiments ranged from 40-6O0/0.Oxidation of Cholesta-5,7',22-trien-3/l-ol (111) and the Extraction of Isovaleric Acid (ZV) Cholesta-5,7,22-trien-3p-o1 (20-25 mg) was dissolved in tert-butanol (90 ml) and to this was added K2C0, (9 ml; 50 mM) and a mixture (50 ml) of KMnO, (3 mM) and NaIO, (97 mM). The mixture was maintained at 37 "C overnight in a gently shaking stoppered flask. Isovaleric acid (5 ml) was then added, and the reaction mixture was acidified with H2S0, and decolourised by the dropwise addition of sodium metabisulphite. The solut.ion was extracted with di-