Oil-bodies, from the immature cotyledons of sunflower (Helianthus annuus L.), were difficult to purify to homogeneity using conventional techniques. The major protein contaminants were albumin and globulin storage proteins. A protocol has been developed, therefore, based upon the stringent washing of the oil-body fraction in 9 M urea, which effectively removed almost all the contaminating protein as judged by SDS/PAGE. The urea-washed oil-bodies were enriched in two major proteins of M(r) 19000 and 20000. These proteins were oleosins as demonstrated by their amino acid compositions and the sequence analysis of peptides produced by CNBr cleavage. Far-UV CD spectra of the oleosins in trifluoroethanol, trifluoroethanol/water mixtures and as mixed micelles in SDS, were typical of alpha-helical proteins with alpha-helical contents of some 55%. The phospholipid content of the urea-washed preparations was less than 0.1% of that required to form a half-unit membrane surrounding the oil-body. The oil-body surface therefore appears to be an unusual and novel structure, covered largely by an oleosin protein coat or pellicle rather than a conventional fluid membrane, half-unit or otherwise.

No abstract

The biosynthesis of C 18 polyunsaturated fatty acids has been studied in the fungus Mucor circinelloides. Microsomal membrane preparations contained ∆9, ∆12 and ∆6 desaturase activities. The ∆9 desaturase exhibited characteristics similar to those of the animal and yeast ∆9 desaturases in being membrane bound and utilising stearoyl-CoA as substrate. Cytochrome b 5 (a soluble form lacking the 20-amino-acid hydrophobic C-terminus) stimulated desaturation and was identified as a major cytochrome component of the membranes. A high ferricyanide reductase activity (indicative of NADH:cytochrome b 5 reductase activity) coupled to inhibition by cyanide further supported the similarity with the mammalian and yeast enzymes. Time-course studies with radiolabelled oleoyl-CoA showed that the oleate [18:1(9)] was transferred to position sn-2 of phosphatidylcholine (PtdCho) and was desaturated to linoleoyl-PtdCho. Removal of the excess oleoyl-CoA from the membranes prior to addition of reductant confirmed that oleoyl-PtdCho is a substrate for the ∆12 desaturase. The entry of oleate at this position of the phospholipid was facilitated by the activity of lyso-PtdCho:acyl-CoA acyltransferase (LPCAT), which readily transferred oleate from oleoyl-CoA to lyso-PtdCho. Desaturation of oleate at the sn-1 position of PtdCho was also demonstrated after the entry of oleate in to the phospholipid by the enzymes of the Kennedy pathway. Thus oleate at sn-1 and sn-2 positions served as substrate for the ∆12 desaturase and is consistent with observations in oil seed tissues. LPCAT activity was substantially higher than that observed with lysophosphatidylethanolamine: acyl-CoA acyltransferase (LPEAT) indicating that oleate is less effectively channelled to phosphatidylethanolamine for linoleate synthesis. No desaturation on phosphatidylinositol could be demonstrated. ∆6 desaturase utilised linoleate at the sn-2 position of exogenously supplied PtdCho presented to the membranes in the presence of reductant. Thus, the entry of substrates into PtdCho via LPCAT and the synthesis of linoleate [18:2(9,12)] and γ-linolenate [18:3(6,9,12)] on this phospholipid is similar to that reported for oil seed membranes.Keywords : Mucor ; oil biosynthesis; polyunsaturated fatty acid ; desaturase substrate; phosphatidylcholine.The biosynthesis of polyunsaturated fatty acids in plants [1Ϫ that contain high levels of 18:3(6,9,12) [12Ϫ14] and have been 3] and animals [4,5] and their assembly to produce storage fats produced on an industrial scale [15]. We have studied previously and oils has been studied extensively. By comparison, and with the biosynthesis of 18:3(6,9,12) in higher plants and characterthe exception of oleaginous yeast [6,7], few detailed biochemi-ised the substrates and stereospecificity of the desaturases incal studies on lipid synthesis in fungi have been undertaken. Of volved in its formation [16Ϫ18]. In borage seed microsomes, particular commercial interest is the production of 'speciality the ∆12 desaturase utilised oleate esterified to positi...

No abstract

TAG-synthesizing capacity was greatest in the microsomal membrane fraction, which accumulated high levels of phosphatidic acid in the presence of glycerol 3-phosphate and acyl-CoA substrates a t pH 7-0. Further metabolism of phosphatidic acid to diacylglycerol and TAG was achieved by increasing the pH to 80. Lysophosphatidic acid : acyl-CoA acyltransferase (LPAAT) activity was particularly high and may have accounted for the rapid accumulation of phosphatidic acid in the membranes. The glycerol-3-phosphate : acyl-CoA acyltransferase (GPAAT) and LPAAT were non-specific for a range of saturated and unsaturated species of acyl-CoA although the GPAAT showed a marked selectivity for palmitoyl-CoA and the LPAAT for oleoyl-and linoleoyl-CoA. y-Linolenic acid was detected a t all three positions of sn-TAG and was particularly enriched a t the sn-3 position. The preparation of active in vitro systems (microsomal membranes) capable of the complete biosynthetic pathway for TAG assembly may be valuable in understanding the assembly of oils in future transgenic applications.