BIOSYNTHESIS OF TRIACYLGLYCEROLS CONTAINING RICINOLEATE IN CASTOR MICROSOMES USING 1-ACYL-2-OLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE AS THE SUBSTRATE OF OLEOYL-12-HYDROXYLASE

We have examined the biosynthetic pathway of triacylglycerols containi ng ricinoleate to determine the steps in the pathway that lead to the high levels of ricinoleate incorporation in castor oil. The biosynthet ic pathway was studied by analysis of products resulting from castor m icrosomal incubation of mitoyl-2-[C-14]oleoyl-sn-glycero-3-phosphochol ine, the substrate of oleoyl-12-hydroxylase, using high-performance li quid chromatography, gas chromatography, mass spectrometry, and/or thi n-layer chromatography. In addition to formation of the immediate and major metabolite, 1-palmitoyl-2-[C-14]ricinoleoyl-sn-glycero-3 -phosph atidylcholine, C-14-labeled 2-linoleoyl-phosphatidylcholine (PC), and C-14-labeled phosphatidylethanolamine were also identified as the meta bolites. In addition, the four triacylglycerols that constitute castor oil, triricinolein, 1,2-diricinoleoyl-3-oleoyl-sn-glycerol, 1,2-diric inoleoyl-3-linoleoyl-sn-glycerol, 1,2-diricinoleoyl-3-linolenoyl-sn-gl ycerol were also identified as labeled metabolites in the incubation a long with labeled fatty acids: ricinoleate, oleate, and linoleate. The conversion of PC to free fatty acids by phospholipase A(2) strongly f avored ricinoleate among the fatty acids on the sn-2 position of PC. A major metabolite, 1-palmitoyl-2-oleoyl-sn-glycerol, was identified as the phospholipase C hydrolyte of the substrate; however, its conversi on to triacylglycerols was blocked. In the separate incubations of 2-[ C-14]ricinoleoyl-PC and [C-14]ricinoleate plus CoA, the metabolites we re free ricinoleate and the same triacylglycerols that result from inc ubation with 2-oleoyl-PC. Our results demonstrate the proposed pathway : 2-oleoyl-PC --> 2-ricinoleoyl-PC --> ricinoleate --> triacylglycerol s. The first two steps as well as the step of diacylglycerol acyltrans ferase show preference for producing ricinoleate and incorporating it in triacylglycerols over oleate and linoleate. Thus, the productions o f these triacylglycerols in this relatively short incubation (30 min), as well as the availability of 2-oleoyl-PC in vivo, reflect the in vi vo drive to produce triricinolein in caster bean.