A highly active soluble diacylglycerol synthesizing system from developing rapeseed, Brassica napus L

Abstract

The subcellular distribution of the enzymes of triacylglycerol biosynthesis has been studied in developing oilseed rape. All in vitro enzymatic activities from oleoyl-CoA to triacylglycerol were sufficient to account for the known rate of oleate deposition in triacylglycerol in vivo. The enzymatic activities from oleoyl-CoA to diacylglycerol preferentially were localized in a 150,000 g supernatant fraction, while the diacylglycerol acyl-transferase mostly was associated with the microsomal (20,000 g pellet and 150,000 g pellet) and oil-body fractions. The soluble (150,000 g supernatant) fraction rapidly incorporated oleate from [1-14C]oleoyl-CoA into diacylglycerol with rates of 40 nm min-1 g-1 FW at 20 microM oleoyl-CoA. The pH optimum was 7.5-9.0, and normal saturation kinetics were seen with oleoyl-CoA; the S0.5 was about 32 microM. Exogenous acyl acceptors, such as glycerol 3-phosphate, lysophosphatidic acid and lysophosphatidyl-choline stimulated oleate incorporation into diacylglycerol. The detergents Triton X-100 and sodium cholate inhibited diacylglycerol formation at concentrations in the region of their critical micellar concentration, while n-octyl-beta, D-glyco-pyranoside had no effect, even at high concentration. The significance of these findings for the mechanism of oil-body formation in developing oilseeds is discussed.