Abstract
The mitochondrial metabolism of unsaturated fatty acids with conjugated double bonds at odd-numbered positions, e.g. 9-cis, 11-trans-octadecadienoic acid, was investigated. These fatty acids are substrates of beta-oxidation in isolated rat liver mitochondria and hence are expected to yield 5,7-dienoyl-CoA intermediates. 5, 7-Decadienoyl-CoA was used to study the degradation of these intermediates. After introduction of a 2-trans-double bond by acyl-CoA dehydrogenase or acyl-CoA oxidase, the resultant 2,5, 7-decatrienoyl-CoA can either continue its pass through the beta-oxidation cycle or be converted by Delta3,Delta2-enoyl-CoA isomerase to 3,5,7-decatrienoyl-CoA. The latter compound was isomerized by a novel enzyme, named Delta3,5,7,Delta2,4, 6-trienoyl-CoA isomerase, to 2,4,6-decatrienoyl-CoA, which is a substrate of 2,4-dienoyl-CoA reductase (Wang, H.-Y. and Schulz, H. (1989) Biochem. J. 264, 47-52) and hence can be completely degraded via beta-oxidation. Delta3,5,7,Delta2,4,6-Trienoyl-CoA isomerase was purified from pig heart to apparent homogeneity and found to be a component enzyme of Delta3,5,Delta2,4-dienoyl-CoA isomerase. Although the direct beta-oxidation of 2,5,7-decatrienoyl-CoA seems to be the major pathway, the degradation via 2,4,6-trienoyl-CoA makes a significant contribution to the total beta-oxidation of this intermediate.
Highlights
The -oxidation of a fatty acid with two conjugated double bonds at odd-numbered positions would produce 5,7-dienoylCoA, which may be dehydrogenated by acyl-CoA dehydrogenase to 2,5,7-trienoyl-CoA
The -oxidation of typical polyunsaturated fatty acids requires the involvement of three auxiliary enzymes in addition to the enzymes that catalyze the four basic reactions of the -oxidation spiral [1]
This result suggests the presence of a mitochondrial pathway for the -oxidation of fatty acids that have odd-numbered conjugated double bonds
Summary
Materials—CoASH, Q-Sepharose, polybuffer exchanger 94, polybuffer 96, reactive red 120, Sepharose CL-6B, phenylmethylsulfonyl fluoride, polyethylene glycol with an average Mr of 8000, benzamidine hydrochloride, acyl-CoA oxidase from Arthrobacter species, Staphylococcus aureus (Cowan strain) suspension (10%, w/v), and all standard biochemicals were obtained from Sigma. (4-Carboxybutyl)triphenylphosphonium bromide, trans-2-pentenal, and lithium bis(trimethylsilyl)amide were purchased from Aldrich. The column was extensively washed with buffer A and eluted with 600 ml of buffer A containing 0.2 M KCl. Fractions with trienoyl-CoA isomerase activity were combined, and the pH was adjusted to 6.3 with 1 M KH2PO4. 1.5 mg of rat liver mitochondria were incubated in 1.9 ml of a basal medium containing 20 mM Tris-HCl (pH 7.4), 4 mM potassium Pi, 0.1 M KCl, 4 mM MgCl2, and 0.1 mM EGTA To this mixture were added, in the indicated orders, bovine serum albumin (0.5 mg/ml), 0.5 mM L-carnitine, 0.5 mM L-malate, 1 mM ADP, and one of the following: 15 M linoleoyl-CoA, 15 M 9-cis,11-transoctadienoyl-CoA, 0.1 mM decanoic acid, or 0.1 mM 5,7-decadienoic acid. After evaporation of acetonitrile under vacuum, the product was concentrated by use of a Sep-Pak C18 cartridge
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