Purification and Physicochemical Properties of Fatty Acid Synthetase and Acetyl‐CoA Carboxylase from Lactating Rabbit Mammary Gland

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Abstract
Fatty acid synthetase was purified 13‐fold from lactating rabbit mammary glands by a procedure which involved chromatography on DEAE‐cellulose, ammonium sulphate precipitation and gel filtration on Sepharose 4B. The preparation was completed within two days and over 100 mg of enzyme was isolated from 100–150g of mammary tissue, which represented a yield of over 40%. The preparation was homogeneous by the criteria of polyacrylamide gel electrophoresis and ultracentrifugal analysis. The sedimentation constant, s20, w was 13.3S, the absorption coefficient, A280nm1%, measured refractometrically was 10.0 ± 0.1, and the amino acid composition was determined. The subunit molecular weight determined by gel electrophoresis in the presence of sodium dodecyl sulphate was 252 000 ± 6 000, and the molecular weight of the native enzyme measured by sedimentation equilibrium was 515 000. These experiments indicate that at the concentrations which exist in mammary tissue (2–4 mg/ml) fatty acid synthetase is a dimer. The purified enzyme did however show a tendency to dissociate to a monomeric 9‐9S species on storage for several days or following exposure to a low ionic strength buffer at pH 8.3. There was only a small quantity of alkali labile phosphate (0.2 molecules per subunit) bound covalently to the purified enzyme. Acetyl‐CoA carboxylase was purified 300‐fold in a 50% yield within 24h by ammonium sulphate and polyethylene glycol precipitations [Hardie, D. G. and Cohen, P. (1978) FEBS Lett. 91, 1–7]. The preparation was in a state approaching homogeneity as judged by polyacrylamide gel electrophoresis, gel filtration on Sepharose 4B and ultracentrifugal analysis. The sedimentation constant, s20, w, was 50.5S, the absorption index, A280nm1%, was 14.5 ± 0.7, and the amino acid composition was determined. The subunit molecular weight of acetyl‐CoA carboxylase determined by gel electrophoresis in the presence of sodium dodecyl sulphate was identical to that of fatty acid synthetase (252 000) as shown by electrophoresis of a mixture of the two proteins. The preparations also contained two minor components of molecular weight 235 000 and 225 000, which appear to be derived from the major species of mol. wt 252 000. A large amount of phosphate (3.2 molecules per subunit) was found to be bound covalently to the purified enzyme. The properties of fatty acid synthetase and acetyl‐CoA carboxylase are compared to those obtained by other workers.