The Isolation and General Properties of Escherichia coli Malonyl Coenzyme A-Acyl Carrier Protein Transacylase

• Published in: The Journal of biological chemistry 1973-12, Vol.248 (23), p.8086-8094
• Main Authors: Ruch, Frank E., Vagelos, P. Roy
• Format: Article
• Language: English
• Subjects: Acyltransferases - analysis; Acyltransferases - isolation & purification; Acyltransferases - metabolism; Amino Acids - analysis; Ammonium Sulfate; Bacterial Proteins; Carbon Radioisotopes; Carrier Proteins; Chromatography, DEAE-Cellulose; Chromatography, Gel; Chromatography, Ion Exchange; Coenzyme A; Dithiothreitol; Electrophoresis, Disc; Electrophoresis, Polyacrylamide Gel; Escherichia coli - enzymology; Evaluation Studies as Topic; Hydrogen-Ion Concentration; Malonates; Methods; Molecular Weight; Oxidation-Reduction; Sodium Dodecyl Sulfate; Sulfhydryl Compounds - metabolism; Ultracentrifugation
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)43197-9

Malonyl coenzyme A-acyl carrier protein transacylase of Escherichia coli was purified 4800-fold by procedures which included chromatography on DEAE-cellulose, Sephadex G-100, Sephadex G-75, DEAE-Sephadex, and preparative polyacrylamide gel electrophoresis. The purified enzyme was shown to be homogeneous by electrophoresis on polyacrylamide gels, by sedimentation equilibrium centrifugation, and by amino acid analysis. A molecular weight of 36,660, obtained with carboxymethylated enzyme by sedimentation equilibrium measurements, agreed with determinations on the native enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (36,500) and by Sephadex G-100 column chromatography (37,000). An s20,w = 2.31 S was determined for the enzyme by sedimentation velocity measurements. Amino acid analysis and determination of the isoelectric point (pH 4.65) both showed the enzyme to be acidic. The purified enzyme was inhibited by N-ethylmaleimide and to a lesser extent by iodoacetamide, but inhibition by both reagents was significantly increased at pH values above 7.3. Preincubation of the enzyme with malonyl-CoA protected against inactivation by both sulfhydryl reagents. Reduction of aged enzyme preparations by incubation with dithiothreitol was shown to stimulate enzyme activity approximately 5-fold. These experiments suggest that a reduced sulfhydryl group(s) on the enzyme is required for maximal catalytic activity.