Separation of the Half-Molecular Weight Nonidentical Subunits of Pigeon Liver Fatty Acid Synthetase by Affinity Chromatography

Two half-molecular weight subunits of 4'-phospho[14C]pantetheine-labeled pigeon liver fatty acid synthetase have been separated and shown to be nonidentical. An affinity chromatographic column containing e-aminocaproylpantetheine bound to cyanogen bromide-activated Sepharose via the e-amino group was used to achieve this separation. The subunit containing 4'-phospho[14C]pantetheine and β-ketoacyl thioester reductase activity is only slightly retarded on this column, whereas the subunit containing acetyl coenzyme A transacylase activity is strongly adsorbed. The latter sub-unit is eluted from the column with 0.1 m phosphate buffer at pH 10. Each subunit is then purified free of small amounts of the other subunit. This is accomplished by subjecting each to conditions which effect fatty acid synthetase formation. Subsequent sucrose density gradient centrifugation then separates the pure half-molecular weight subunits containing transacylase or β-ketoacyl thioester reductase activity from the small amount of fatty acid synthetase complex. Recombination of the purified subunits under appropriate conditions yields enzymatically active fatty acid synthetase complex.