Acetyl CoA carboxylase. I. Requirement for two protein fractions.

E is a biotin enzyme which is carboxylated in reaction (1). In reaction (2) the carboxyl group is transferred to acetyl CoA to form malonyl CoA. Liver acetyl CoA carboxylase has been isolated as a homogeneous protein.26 The preparation with the highest enzymatic specific activity contains 1 mole of biotin per 409,000 gm of protein.4 Ryder et al.4 have reported preliminary investigations which indicate that the protomeric unit is composed of four polypeptide chains of approximately 100,000 mol wt. Thus it is apparent that this carboxylase is composed of nonidentical subunits, only one of them containing biotin, and this is consistent with previous studies of other biotin enzymes."' 1' Studies of the mechanism of biotin enzyme reactions have been hampered by the irreversible inactivation of the enzyme brought about by those treatments which dissociate the protein into its subunits. This communication describes the acetyl CoA carboxylase of Escherichia coli. Routine protein fractionation procedures lead to the separation of two protein fractions required for the over-all reaction. One of these, Ea, contains biotin and is carboxylated to form Ea-CO2as in reaction (1). The other fraction, EB, catalyzes carboxyl transfer from Ea-CO2to acetyl CoA to form malonyl CoA. Materials.-E. coli B, 1/4 log cells were obtained from Grain Processing Corporation. Lactobacillus plantarum 8014 was obtained from the American Type Culture Collection. Sodium C'4-bicarbonate was obtained from New England Nuclear. ATP and CoA were purchased from P-L Laboratories. Avidin was obtained from Nutritional Biochemical Company and alumina COy from Sigma. Acetyl CoA was synthesized as described.'4 Methods.-Preparation of enzyme fractions: Cell-free extracts of E. coli B were prepared by homogenization in a Manton-Gaulin submicron disperser. After removal of cellular debris by centrifugation, nucleic acids were precipitated with MnCl2 and the enzyme was subsequently precipitated between 25 and 45% ammonium sulfate saturation. Attempts at further purification using alumina Cy gel led to the complete loss of enzymatic activity which could, however, be recovered by recombination of two fractions. A summary of this separation is shown in Table 1 where it is seen that one fraction, Eb, was not adsorbed to the gel and remained in the supernatant, whereas Ea was adsorbed to the gel an I was recovered by elution with potassium phosphate, pH 7.7, between 0.1