RICINOLEIC ACID BIOSYNTHESIS AND TRIACYLGLYCEROL ASSEMBLY IN MICROSOMAL PREPARATIONS FROM DEVELOPING CASTOR-BEAN (RICINUS-COMMUNIS) ENDOSPERM

Microsomal membrane preparations from the developing endosperm of castor bean (Ricinus communis) catalysed the transfer of oleate from [C-14]oleoyl-CoA to phosphatidylcholine (PtdCho). In the presence of NADH, radioactive ricinoleate (12-hydroxyoctadec-9-enoate) was synthesized from [C-14]oleate, and this was largely recovered in PtdCho and as free fatty acid. The addition of unlabelled ricinoleoyl-CoA to these incubation mixtures did not increase the low [C-14]ricinoleate concentration found in the acyl-CoA fraction nor decrease the [C-14]ricinoleate concentration in PtdCho and free fatty acid, and thus no evidence was obtained for a hydroxylation with oleoyl-CoA as a substrate. The addition of NADH, necessary for the formation of ricinoleate, caused a decrease of the total radioactivity in PtdCho with a corresponding increase in the amount of label in free ricinoleic acid. This increase was due to the action of a phospholipase A, which released ricinoleic acid but not oleic acid from PtdCho. Such a phospholipase activity, attacking ricinoleoyl-PtdCho but not oleoyl-PtdCho, was also demonstrated in microsomal preparations from developing cotyledons of safflower and oil-seed rape. An analysis of the acyl groups at different positions in microsomal PtdCho of castor bean showed that ricinoleate was almost entirely associated with position sn-2. Likewise the [C-14]ricinoleate in [C-14]PtdCho formed after incubations with microsomal preparations with NADH and [C-14]oleoyl-CoA resided in position sn-2 with none in position sn-1. In contrast, the [C-14]linoleate formed by desaturation of [C-14]oleoyl-PtdCho was present at both positions. In the presence of ATP, CoA and Mg2+, the ricinoleate acid released from PtdCho was activated to ricinoleoyl-CoA. The ricinoleoyl-CoA was an efficient acyl donor in the acylation of glycerol 3-phosphate (Gro3P) to yield phosphatidic acid and triacylglycerols. In microsomal preparations incubated with an equimolar mixture of [C-14]oleoyl-CoA and [C-14]ricinoleoyl-CoA in the presence of Gro3P, only a minor amount of [C-14]ricinoleate entered PtdCho, and this was believed to be via the exchange of phosphocholine groups between a diacylglycerol pool and the PtdCho. On the basis of our results, a scheme of ricinoleate formation and its incorporation into triacylglycerols in castor-bean endosperm is proposed.