Abstract
1. 1. The crystallization of thiolase specific for long-chain β-keto-CoA derivatives (acyl-CoA:acetyl-CoA C-acyltransferase, EC 2.3.1.16) from ox liver is described. The enzyme cleaves β-keto-CoA derivatives with a chain-length of 4–16 C atoms. Optimal activity of the enzyme was found with β-ketocapronyl-CoA. 2. 2. The purification of enoyl-CoA reductase (NADPH: enoyl-CoA oxidoreductase) from rat liver is described. Separation of thiolase and the greater part of β-hydroxyacyl-CoA dehydrogenase ( l-3-hydroxyacyl-CoA:NAD + oxidoreductase, EC 1.1.1.35) from enoyl-CoA reductase was achieved by treatment of microsomes with sodium cholate and subsequent fractionation of the particles in the ultracentrifuge. Enoyl-CoA reductase is specific for the α,β-unsaturated CoA derivatives with chain lengths of 4–16 C atoms. Optimal activity of the enzyme was found with 2,3-hexenyl-CoA. 3. 3. Combination of purified thiolase, β-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase ( l-3-hydroxyacyl-CoA hydro-lyase, EC 4.2.1.17) and enoyl-CoA reductase with [ 14C]acetyl-CoA-, NADPH- and NADH-regenerating systems resulted in the presence of longchain acyl-CoA derivatives and in the elongation of the latter by 1–2 C 2 units. Avidin had no influence on the incorporation of radioactive acetate. The intermediate formation of malonyl-CoA as a reaction partner of the long-chain acyl-CoA derivatives was thus excluded. 4. 4. The physiological significance of this reaction sequence and its relation to the malonyl-CoA-independent fatty acid synthesis and chain elongation of fatty acids reported by various authors to occur in mitochondria is discussed.
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More From: Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism
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