A new assay procedure to study the induction of β-ketoacyl-ACP synthase I and II, and the complete purification of β-ketoacyl-ACP synthase I from developing seeds of oilseed rape ( Brassica napus)

Abstract

We have developed a new radiochemical assay for β-ketoacyl-acyl carrier protein synthase, which is rapid, sensitive and does not require the presynthesis of acyl-acyl carrier protein derivatives as substrates. The assay has allowed us to study the induction of β-ketoacyl-acyl carrier protein synthase I and II activities during development of seeds of oilseed rape ( Brassica napus). Both enzymes are coordinately induced prior to lipid accumulation in these seeds. We have also used the new assay to purify to homogeneity β-ketoacyl-acyl carrier protein synthase I from the developing seeds. The enzyme required an approx. 12000-fold purification from the cell-free crude extract, with 8 μg of enzyme being obtained from 300 g of seed. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis purified β-ketoacyl-acyl carrier protein synthase I migrated as a single polypeptide giving an M r value of 43000. Gel-filtration chromatography gave an apparent native M r value of 86 700, suggesting that the enzyme is a homodimer. On electrophoresis in non-denaturing gels, two polypeptide bands were detectable, and both were associated with β-ketoacyl-acyl carrier protein synthase I activity. On gel filtration of a partially purified preparation, β-ketoacyl-acyl carrier protein synthase I and II could be partially resolved, with synthase II having an apparent native M r value of 87 400. The two activities could be completely resolved on an FPLC Mono Q ion-exchange column.