[12] Acyl carrier protein synthetase

Publisher Summary This chapter discusses the determination of acyl carrier protein (ACP) synthetase. The conversion of apo-ACP to ACP is measured in a two-stage assay. In the first-stage synthesis of ACP is allowed to proceed, catalyzed by ACP synthetase. This stage is terminated, and the amount of ACP synthesized is determined in the second stage by means of the malonyl-CoA-CO2 exchange reaction. Apo-ACP can be prepared enzymatically from ACP or chemically using 60% HF to cleave the phosphodiester bond linking the hydroxyl of serine-36 to 4´-phosphopantetheine. In the course of purification, all operations are carried out at 4°. Escherichia coli strain B are thawed and suspended in 2 liters of 0.01 M Tris-HCl, pH 7.4 containing 0.01 M 2-mercaptoethanol and 0.0001 M ethylenediaminetetraacetic acid (EDTA). The suspension is ruptured in a Manton–Gaulin submicron dispersor at 9000 psi. To the viscous stirred suspension is added 800 ml of a 10% streptomycin sulfate solution and the suspension is centrifuged for 30 minutes at 27,500 g. The supernate is saved (3400 ml, 55 g protein).