Purification and Physicochemical Properties of ATP Citrate ( pro‐3S ) Lyase from Lactating Rat Mammary Gland and Studies of Its Reversible Phosphorylation

A simple procedure is described for the isolation of ATP‐citrate lyase, acetyl‐CoA carboxylase and fatty acid synthase from lactating rat mammary gland. ATP‐citrate lyase was purified 48‐fold by poly(ethyleneglycol) precipitation, ammonium sulphate precipitation and chromatography on DEAE‐Sepharose at pH 7.5. The preparation was completed in 2–3 days and 30 mg of enzyme was isolated from 10 animals, which represented a yield of 38%. The preparation was of a high degree of purity as judged by polyacrylamide gel electrophoresis and ultracentrifugal analysis. The sedimentation constant, S 20,w was 13.6 ± 0.2 S, the absorption coefficient, A 1% 280nm measured refractometrically was 12.2 ± 0.7 and the amino acid composition was determined. The subunit molecular weight determined by gel electrophoresis in the presence of sodium dodecyl sulphate was 116000 and the molecular weight of the native enzyme measured by sedimentation equilibrium was 473000. These experiments indicate that mammary ATP‐citrate lyase is a tetramer. The purified enzyme contained 0.5 mol of alkali‐labile phosphate covalently bound/subunit. The phosphorylation of ATP‐citrate lyase by cyclic‐AMP‐dependent protein kinase reached a plateau when 0.7 ± 0.2 mol of phosphate were bound to a serine residue in a single tryptic peptide. The same tryptic peptide was phosphorylated by cyclic‐GMP‐dependent protein kinase, although at a much slower rate. ATP‐citrate lyase was not phosphorylated by phosphorylase kinase or by glycogen synthase kinase 3. The phosphate introduced by cyclic‐AMP‐dependent protein kinase could be completely removed by incubation with protein phosphatase 1 or protein phosphatase 2. However the alkali‐labile phosphate present in ATP‐citrate lyase as isolated was not removed by these protein phosphatases, indicating that it is located at a distinct site(s). Phosphorylation of purified ATP‐citrate lyase by cyclic‐AMP‐dependent protein kinase did not alter the V or the apparent K m for citrate, ATP or CoA. The properties of mammary ATP‐citrate lyase are compared to those of the rat liver enzyme and the possible role of phosphorylation in the regulation of its activity is discussed.