The β‐Oxidative Cleavage of Long‐Chain Fatty Acids in Rat‐Liver Cytoplasm

Abstract

A β‐oxidative cleavage of oleyl‐ and stearyl‐CoA has been obtained with soluble enzymes of rat‐liver homogenate. The transformation requires NAD+ as an oxido‐reductive cofactor and is enhanced by ATP and CoA. Under the reported conditions 2,9‐octadecadienoic, 3,9‐octadecadienoic, 3‐hydroxy‐9‐octadecenoic, 7‐hexadecenoic, 3‐hydroxy‐7‐hexadecenoic and 5‐tetradecenoic acids are present after incubation of oleyl‐CoA, while no detectable amounts of shorter fatty acids are formed. A similar oxidation pattern is obtained with stearyl‐CoA when Mg2+ ions are also added to the system. The cytoplasmic origin of these enzymes is established by comparing their activity with that of the mitochondrial β‐oxidation enzymes.

Studies on the 14α‐Demethylation Mechanism in Cholesterol Biosynthesis

“…Sa-Cholest-7-en-3-one [22] (500 mg) in CH302H (99.5% isotopic enrichment, 15 ml) was refluxed under nitrogen for 4 h in the presence of sodium methoxide. Deuterated methanol was distilled and the exchange procedure was repeated twice.…”

“…Crystallization of the residue from deuterated methanol yielded pure (2,3-2H4)cholest-7-en-3-one (480 mg), m.p. 146-147 "C, [a]: = 24.9" (c = 1 in CHC13) [22]. The mass spectrum of the compound showed the molecular ion at mje 388.…”

“…5a-(2,4-2H4)Cholest-7-en-3/?-ol (350 mg) was obtained from the 3-ketone as described previously [22]. The molecular ion of the trimethylsilyl derivative of the compound was at mje 462 and only traces of the protium species at m/e 458 were detectable.…”

“…The molecular ion of the trimethylsilyl derivative of the compound was at mje 462 and only traces of the protium species at m/e 458 were detectable. The following tetradeuterated sterols were prepared either from this compound or from its acetate derivative obtained by treatment with acetic anhydride in pyridine ( M + at m/e 432, tR 1.1 8), using previously described methods; 5a-(2,4-2H4)cholest-8 (14) [22] ; 5a-(2,4-2H4)cholest-7-ene-3P,14a-diol (Me&-: M + at m/e 460, tR 1.02, acetate: M' at m/e 430, tR 1.0) was prepared as described above for the non-deuterated compound using as starting material 3P-acetylo~y-8a,l4a-epoxy-5a-(2,4-~H~) cholestan-7-one prepared from the acetate of 5~-(2,4-~H4)cholest-7-en-3P-o1 [17]. The identity and purity of all the tetradeuterated compounds was checked by comparing their chemico-physical properties with those of authentic standards.…”

“…Absence of day light was also maintained during the extraction of the incubation mixtures and their preparation for analysis. Sterol substrates were added to the incubation mixture as suspensions in 0.1 M phosphate buffer pH 7.4, 30 mM nicotinamide, 4 mM MgC12 and containing Tween 80 [22] to a final concentration of 5 mg/ml in the incubation mixture [12]. The same concentration of Tween 80 was added to incubations with [3H]formate as the substrate.…”