10] Purification of plant acetyl-CoA: Acyl carrier protein transacylase

Abstract

This chapter describes the assay, purification, and properties of plant acetyl-CoA: acyl carrier protein transacylase. Acetyl-CoA: acyl carrier protein transacylase (acetyl transacylase) catalyzes the transfer of the acetyl group from the SH group of CoA to that of acyl carrier protein (ACP). The Escherichia coli and plant acetyl transacylases can be easily separated from the other component enzymes of fatty acid synthetases (FAS) by conventional purification procedures. Acetyl transacylase is assayed by counting the radioactivity of [1- l4 C]acetate transferred to ACP from CoA. The reaction is stopped by addition of cold perchloric acid and the mixture is poured on a Millipore filter. After unreacted [ 14 C]acetyl-CoA is washed out with cold perchloric acid, the filter containing acid-insoluble [ 14 C]acetyl-ACP is transferred to a scintillation vial and counted to determine the amount of acetyl-ACP present. The molecular weight of the purified acetyl transacylase is estimated to be 48,000 by Sephacryl S-300 gel filtrations. The acetyl transacylase is maximally active at pH 8.1, with the half-maximum activity at pH 7.5, and about 78% of the maximum activity is retained at pH 9.1.