Purification and characterization of maize leaf acetyl-coenzyme A carboxylase.

Maize leaf acetyl-CoA carboxylase was purified from whole tissue homogenates by precipitation with polyethylene glycol and ammonium sulfate, and gel filtration. Recoveries were approximately 5% with 100-fold increases in specific activity. The molecular weight of the native enzyme is estimated at 500,000 from the elution volume of a calibrated Ultrogel AcA 22 column. Electrophoresis in polyacrylamide gel containing 1% sodium dodecyl sulfate revealed a single subunit of Mr 60,000-61,000. Investigation of the kinetic properties of the purified enzyme indicates that Mg X ATP is the active substrate, with free ATP inhibiting and Mg2+ activating the enzyme. Km's for acetyl-CoA and HCO3- are about 0.1 and 2 mM, respectively. ADP inhibition is competitive with respect to ATP, but uncompetitive with respect to acetyl-CoA. The observed responses of purified acetyl-CoA carboxylase to changes in pH, and in concentrations of Mg2+, ATP, and ADP, and the reported changes in the chloroplastic concentrations of these effectors during light-dark transitions of chloroplasts are consistent with increased acetyl-CoA carboxylase activity upon illumination of chloroplasts.