Expression and properties of acyl-CoA binding protein from Brassica napus

Expression and characteristics of an acyl-CoA binding protein (ACBP) from Brassica napus L. were examined. A cDNA encoding an ACBP from rape was over-expressed in E. coli and the resulting protein (rACBP) was purified by ammonium sulphate precipitation followed by gel filtration chromatography. SDS-PAGE showed the protein to be greater than 99 % pure and to have a molecular weight of approximately 10 kDa. Lipidex-1000 competition assays showed that the rACBP purified by this method is fully functional, binding both oleoyl- and palmitoyl-CoA in the ratio 1 mol protein:1 mol acyl-CoA. Native isoelectric focusing revealed the presence of two isoforms of rACBP in ammonium sulphate and gel-filtration purified preparations. These isoforms were separated by chromatofocusing and shown to differ in mass by 131 Da, the mass of one methionine residue. Both of these isoforms bound palmitoyl-CoA with similar affinities. Antibodies raised to purified rACBP were used to study tissue specific and developmental expression of ACBP. Western blots revealed the presence of ACBP in all tissues examined and the level of expression was similar in many of those tissues. The amount of ACBP was not, however, strongly correlated with rates of lipid biosynthesis during embryo development nor with lipid degradation during seedling germination. The addition of rACBP stimulated microsomal glycerol-3-phosphate acyl-transferase (GPAT; EC 2.3.1.15) activity in in vitro assays, but concentrations in excess of a 1:1 ratio of ACBP:oleoyl-CoA caused decreases in GPAT activity.