Labelled oleic acid, which is used as a substrate in studying the conversion of oleate into linoleate by Chlorella chloroplasts, is rapidly incorporated into the phospholipids. The phosphatidyl choline fraction accounts for almost all of the phospholipid label. Desaturation to linoleate lags behind the incorporation into lipids, but the newly synthesized linoleate remains esterified to phosphatidyl choline.[1-14C]Oleoy1-phosphatidyl choline, incubated with chloroplasts, is converted into [1-14C]-linoleoyl-phosphatidyl choline, but considerable difficulties arise in presenting the lipid to the enzyme in an acceptable form. Oleoyl-phosphatidyl choline formed in situ, however, does not present these difficulties and is desaturated a t a faster rate than the CoA derivative under conditions in which unesterified fatty acid cannot be LLactivated", and hence cannot be desatmated. We conclude that the precursor fatty acid can be transferred directly from the lipid to the desaturase enzyme and the product immediately accepted by a lipid to form a "desaturationacylation" cycle. Our data neither prove, nor exclude the possibility that the fatty acid is transformed while still attached to the lipid in ester linkage. esters are not the true substrates for the "desaturase". The work of Nagai and Bloch [6] with the stearoylacyl carrier protein desaturase of Euglena gracilis might suggest that the acyl carrier protein ester is the true substrate. However, the experiments of Nichols and James [7,8] have pointed to the possibility that phospholipids and glycolipids may have an important role to play in fatty acid transformations, while, more specifically, Harris et al. 141 have speculated about the possibility that phosphatidyl choline, which has a striklingly high specific activity with respect to newly biosynthesized linoleate in Chlorelh, may have special importance for the desaturation reaction.I n this paper, we shall report further studies which confirm that phosphatidyl choline is an important intermediate in the conversion of oleate into linoleate. MATERIALS AND METHODSMost of the procedures have been documented in an earlier paper [3]. However, some additional techniques have been used. Thin Layer ChromatographyTotal lipids were separated on thin layers (0.25 mm) of Silica Gel G with the solvent system:

1. Crambe seeds harvested 6 -30 days after flowering actively incorporated radioactive precursors into lipids. The distribution of label among the different lipid classes depended on the precursor, the age of the seed, and the duration of the incubation.2. Labelled acetate was incorporated preferentially into phospholipids of whole seeds but the proportion of the radioactivity located in triglycerides increased as the age of the seed increased.3. Uniformly labelled glucose was incorporated readily into the glycerol moiety of glycerolipids but not a t all into the fatty acid moiety. The largest proportion was found in the phospholipid fraction and this proportion increased as the age of the seed increased, in contrast to labelled acetate.4. 1:nlformly labelled glycerol was actively incorporated into glycerolipids of whole seeds. Phospholipids were the first fraction to be labelled, and in this fraction labelled phosphatidic acid could be detected at early time periods. Subsequently, diglycerides became labelled and finally triglycerides. Labelled glycerol was quickly converted into a number of water-soluble metabolites, one of the most rapidly labelled of which was glycerol phosphate. Taken together these data are consistent with the presence of the glycerol phosphate pathway for triglyceride synthesis in maturing Crambe seeds.5. A fat fraction has been isolated from mature and maturing Crambe seeds by homogenisation of the seeds followed by flotation in the centrifugc. Electron microscopic examination indicated that the fraction contains the oil bodies that are the storage form of the oil in the seed in vivo.6. The isolated oil bodies contain 75O/, by weight of lipid and 190/, of protein. About 900/, of the lipid is triglyceride, the remainder is mostly phospholipid. No glycolipid is present. .The isolated fat fraction catalysed the conversion of different substrates, malonyl-CoA, long chain acyl-CoA and glycerol 3-phosphate into lipids. Evidence is presented that this is not due to contamination from other cell fractions.The pathways of biosynthesis of triglycerides are well worked out in animal tissues [l]. In plants very little published information exists on the overall pathways by which triglycerides arise, even less on the detailed enzymic steps. Labelled glycerol 3-phosphate is incorporated into phosphatidic acid by spinach-leaf microsomal fractions [2,3] ___-phate first into phosphatidic acid, then into diglycerides and finally into triglycerides, but no similar work has been done with seeds.In a previous paper we have described changes in oil composition as the Crambe seed matures and the complete stereospecific analysis of the seed-oil triglycerides [ 5 ] . Electron microscopy of thin sections of mature Crambe seeds, fixed and stained with osmium tetroxide, revealed cells packed with oil bodies with a mean diameter of about 1 pm. I n this paper we describe the isolation of the oil storage bodies, their chemical composition, and their enzymic activity and present evidence for the existence of the glyce...

Hen liver microsomal stearoyl-CoA desaturase has been prepared in "soluble" form, either by extraction of freeze-dried microsomal fractions with high ionic strength phosphate buffer or by treatment with non-ionic detergents. On a discontinuous density gradient, desaturase activity was recovered in the lightest fraction which is rich in lipids. On agarose gel, the desaturase activity was excluded in the void volume and therefore had a particle weight greater than 4 x 106.Removal of some of the lipids of the microsomal fraction led t o inability to solubilize the desaturase, but resulted in enhancement of enzymic activity. Complete removal of lipids was not possible with acetone-water mixtures and preparations from which the maximum amount of lipid had been removed contained one quarter to one half of the original enzymic activity. This residual activity could not be restored to the original value by addition of micellar dispersions of lipids.We have recently reported the solubilization of stearoyl-CoA desaturase (hereafter called desaturase) from a rat liver microsomal fraction [l]. The presence of very active acyl-CoA hydrolases in rat liver presents difficulties in working with acyl-CoA thiol esters and in further work we used hen liver as starting material. This paper describes initial attempts t o purify the hen liver desaturase which have resulted so far in a 4-to 5-fold increase in specific activity. MATERIALS AND illETHODS ReagentsRadiochemicals were purchased from the Radiochemical Centre, Amersham. Coenzyme A (CoA), NADH, NADPH and phospholipase A were products of Sigma (London) Ltd. Triton X-100 (Rohm and Haas, supplied by BDH), and Lubrols 14 and 18 (ICI) were made up as loo/, (w/v) solutions in water.Sodium dodecyl sulphate and cetyl pyridinium chloride both supplied by BDH were made up as 4OlO (w/v) solutions. Acetone was stored over anhydrous K,CO, and distilled over anhydrous K,CO, before use. All other solvents and reagents were of analytical grade and used without further purification. Sephadex and sepharose gels were obtained from Pharmacia, London. Analytical MethodsThe assay of microsomal desaturase El], degradation of phospholipids with phospholipase A [2], and . We had no data on the diet of hens delivered to the slaughterhouse. Birds were usually 1-2 years old; the livers weighed on average 40 g and usually contained large amounts of fat. All steps in the preparation of the microsomal fraction were done at 0-4". The livers were minced and homogenized in 1.5 volumes of 0.3 M sucrose (pH 7.5 adjusted with KOH) for 30sec in a n MSE 'Atomix'. The homogenate was centrifuged a t 1000 x g for 15 min; the supernatant was recentrifuged as before. Mitochondria were sedimented at 15000xg for 20min and the microsomal pellet obtained at 100000 xg for 60 min. At each centrifugation step, a thick layer of yellow triglyceride floated at the surface and was removed with a spatula or by filtration through fine muslin. The red gelatinous pellets from 2-3 kg liver were usually stored in 200 mg batches at -20" ...

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