Allan E. Stafford

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 containing ricinoleate to determine the steps in the pathway that lead to the high levels of ricinoleate incorporation in castor oil. The biosynthetic pathway was studied by analysis of products resulting from castor microsomal incubation of 1-palmitoyl-2-[14C]oleoyl-sn-glycero-3-phosphocholine, the substrate of oleoyl-12-hydroxylase, using high-performance liquid chromatography, gas chromatography, mass spectrometry, and/or thin-layer chromatography. In addition to formation of the immediate and major metabolite, 1-palmitoyl-2-[14C]rici-noleoyl-sn-glycero-3-phosphocholine, 14C-labeled 2-linoleoyl-phosphatidylcholine (PC), and 14C-labeled phosphatidylethanolamine were also identified as the metabolites. In addition, the four triacylglycerols that constitute castor oil, triricinolein, 1,2-diricinoleoyl-3-oleoyl-sn-glycerol, 1,2-diricinoleoyl-3-linoleoyl-sn-glycerol, 1,2-diricinoleoyl-3-linolenoyl-sn-glycerol, were also identified as labeled metabolites in the incubation along with labeled fatty acids: ricinoleate, oleate, and linoleate. The conversion of PC to free fatty acids by phospholipase A2 strongly favored 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 conversion to triacylglycerols was blocked. In the separate incubations of 2-[14C]ricinoleoyl-PC and [14C]ricinoleate plus CoA, the metabolites were free ricinoleate and the same triacylglycerols that result from incubation with 2-oleoyl-PC. Our results demonstrate the proposed pathway: 2-oleoyl-PC. Out results demonstrate the proposed pathway: 2-oleoyl-PC→2-ricinoleoyl-PC→ricinoleate →triacylglycerols. The first two steps as well as the step of diacylglycerol acyltransferase show preference for producing ricinoleate and incorporating it in triacylglycerols over oleate and linoleate. Thus, the productions of these triacylglycerols in this relatively short incubation (30 min), as well as the availability of 2-oleoyl-PC in vivo, reflect the in vivo drive to produce triricinolein in castor bean.

Gradietn C18 high-performance liquid chromatography of gibberelins

Retention times of 66 gibberellins (GA) in gradient C18 high-performance liquid chromatograhy (HPLC) are reported. These include the retention times of 21 new GAs added to our previously reported 24 GAs. Retention of the other 21 GAs are the ranges estimated from previously reported C8 HPLC. The polarity order (elution order) with hydroxyl groups at different locations and orientations is 12α > 13 > 11β > 16α > 1α > 2α > 15β > 1β > 2β > 3α > 3β.

Pathways for fatty acid elongation and desaturation in Neurospora crassa.

Neurospora crassa incorporated exogenous deuterated palmitate (16∶0) and 14C-labeled oleate (18∶1Δ9) into cell lipids. Of the exogenous 18∶1Δ9 incorporated, 59% was desaturated to 18∶2Δ9,12 and 18∶3Δ9,12,15. Of the exogenous 16∶0 incorporated, 20% was elongated to 18∶0, while 37% was elongated and desaturated into 18∶1Δ9, 18∶2Δ9,12, and 18∶3Δ9,12,15. The mass of unsaturated fatty acids in phospholipid and triacylglycerol is 12 times greater than the mass of 18∶0. Deuterium label incorporation in unsaturated fatty acids is only twofold greater than in 18∶0, indicating a sixfold preferential use of 16∶0 for saturated fatty acid synthesis. These results indicate that the release of 16∶0 from fatty acid synthase is a key control point that influences fatty acid composition in Neurospora.

Conversion of palmitate to unsaturated fatty acids differs in a Neurospora crassa mutant with impaired fatty acid synthase activity

The Neurospora crassa cel (fatty acid chain elongation) mutant has impaired fatty acid synthase activity. The cel mutant requires exogenous 16:0 for growth and converts 16:0 to other fatty acids. In contrast to wild-type N. crassa, which converted only 42% of the exogenous [7,7,8,8-2H4]16:0 that was incorporated into cell lipids to unsaturated fatty acids, cel converted 72%. In addition, cel contains higher levels of 18:3δ9,12,15 than wild-type, and synthesizes two fatty acids, 20:2δ11,14 and 20:3δ11,14,17, found at only trace levels in wild-type. Thus, the Δ15-desaturase activity and elongation activity on 18-carbon polyunsaturated fatty acids are higher for cel than wild-type. This altered metabolism of exogenous 16:0 may be directly due to impaired flux through the endogenous fatty acid biosynthetic pathway, or may result from altered regulation of the synthesis of unsaturated fatty acids in the mutant.

Reevaluation of the neutral lipids ofTilletia controversa andTilletia tritici

Differential scanning calorimetry of whole teliospores and lipid extracts ofTilletia controversa Kühn andT. tritica Tul. indicated that the lipid composition of teliospores was different than earlier reported. An exothermic peak at −40 to −45°C and an endotherm at −25 to −15°C indicated that the majority of lipids were triacylglycerols (TAG). Hot isopropanol was used to inactivate lipases during lipid extraction. Thin-layer chromatographic analysis of extracted lipids showed that free fatty acids (FFA) were not present in great quantities unless water was present during lipid extraction. As measured by gas chromatography. FFA accounted for 1–5% of the lipid content in teliospores ofTilletia spp. The TAG content of teliospores was 60–80% of total lipids.