Fatty acid biosynthesis V. Purification and characterisation of fatty acid synthetase from lactating-rabbit mammary gland

Abstract

1. 1. Fatty acid synthetase has been isolated from the particle-free supernatant fraction of lactating-rabbit mammary gland homogenates. 2. 2. Fatty acid synthetase was homogeneous based on the criteria of sedimentation as a single symmetrical peak on analytical ultracentrifugation and elution as a single peak with constant specific activity from Sephadex G-200 and DEAE-52 cellulose. However, traces of acetyl-CoA: COa ligase (ADP) and ATP: citrate lyase were associated with the synthetase. Malonyl-CoA carboxy-lyase activity, for which a modified assay is described, co-purified with fatty acid synthetase. 3. 3. The molecular weight of the fatty acid synthetase was 9.1·10 5 and its hydrated radius 120 Å. It contained 58–59 sulphydryl groups per mole and the amino acid composition is given. 4. 4. The stability of fatty acid synthetase is described. The schlieren patterns of aged preparations showed that dissociation had occurred and this was not reversed on partial reactivation of the enzyme. 5. 5. In the absence of NADPH, fatty acid synthetase formed triacetic lactone from both acetyl-CoA and from acetyl-CoA plus malonyl-CoA. 6. 6. Using optimum substrate concentrations, fatty acid synthetase synthesised a wide range (C 4:0−C 18:0) of fatty acids. The major product was C 16:0 and a significant amount of C 4:0 was also synthesised.