Alterations in the activities of enoyl-CoA hydratase, 3-hydroxyacyl-CoA epimerase, and 2,4-dienoyl-CoA reductase in rat liver mitochondria and peroxisomes caused by clofibrate were compared in order to clarify the metabolic pathways for unsaturated fatty acids. Our results suggest that there are two pathways for fatty acids having (a) double bond(s) at the even-numbered carbon atom(s) both in mitochondria and in peroxisomes. One is the hydratase-epimerase participating pathways, and the other is the reductase participating pathway. It is considered that the former in mitochondria occurs mainly under normal conditions, and the latter becomes significant when the degradation of fatty acids is stimulated.
2,4-Dienoyl-CoA reductase has been detected in crude extracts of E. coli. The reductase was shown to be induced many fold when the cells were grown in the presence of linoleic or oleic acid. The activity profile of the reductase on gel filtration was different from those of other enoyl reductases. These results suggest that there are two pathways even in E. coli for the degradation of cis-4-decenoyl-CoA, which is an intermediate in the beta-oxidation of linoleic acid, as recently proposed in rat liver.
NADPH-dependent trans-2-enoyl-CoA reductase was purified to homogeneity from Escherichia coli. The molecular weight of the native enzyme was estimated to be 80,000 by gel filtration and that of the subunit was estimated to be 40,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was most active toward trans-2-hexenoyl-CoA and trans-2-octenoyl-CoA but was less active toward longer chain substrates, whereas the Km values decreased progressively with increasing carbon chain length of substrates. The reductase appears to have a functional thiol group. The enzyme activity was rapidly decreased by p-hydroxymercuribenzoate and 5,5'-dithiobis(2-nitrobenzoic acid), and slowly by N-ethylmaleimide. The enzyme was protected from inhibition by these SH-reagents by the addition of NADPH. The enzyme was also inhibited by saturated acyl-CoA esters.
2,4-Dienoyl-CoA reductase has been separated from Escherichia coli grown in the presence of linoleic acid and purified to homogeneity. The enzyme has a molecular weight close to 50,000 as determined by gel filtration on Sephacryl S-200 Super-fine. The reductase was rather stable in a buffer containing citric acid and kept its full activity on heating at 55 degrees C for 10 min in the pH range of 5.5 to 6.5, but was completely inactivated on heating at 58 degrees C for 10 min. Phosphocellulose column chromatography revealed that the reductase was not involved in the multi-enzyme complex (molecular weight of 260,000) of fatty acid oxidation.
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