Diacylglycerol acyltransferase in maturing sunflower seeds.

Developing sunflower seeds exhibit a high diacylglycerol acyltransferase (DAGAT, EC 2.3.1.20) activity. The distribution of the enzyme has been studied in subcellular fractions prepared by differential centrifugation of seed homogenate. Its activity was characterized using [1-(14)C]oleoyl-CoA and diolein dispersed in Tween 20. Some properties of the microsomal fraction of DAGAT were investigated. Hyperbolic kinetics were observed, the apparent K(m) was 60 microM and the specific activity of the reaction 15 pmol/min/mg of protein. Addition of BSA (0.1%) stimulated oleate incorporation, which was not dependent on the presence of exogenous diacylglycerol. Detergents which might solubilize DAGAT, Triton X-100 and CHAPS, were tested for enzyme inhibition, and CHAPS was found to be the least denaturing.

Compared selectivities of the phosphatidylinositol-synthase from maize coleoptiles either in microsomal membranes or after solubilization.

PI-synthase selectivity from etiolated maize coleptiles was studied either associated with the microsomal membranes or after solubilization by CHAPS and prepurification on a DEAE-trisacryl M column. When maize microsomes were incubated with [3H]inositol without any exogenous CPM-PA, the most heavily labelled molecular species were 16:0/18:2-PI (77%), 16:0/18:3-plus 18:2/18:2-PI (15%), 16:0/18:1-PI (4%) and 18:0/18:2-PI (4%). Addition to the incubation medium of up to 300 microM 16:0/16:0-CMP-PA unexpectedly resulted in the formation of very little labelled 16:0/16:0-PI. When the solubilized fraction from microsomes was incubated with [3H]inositol in absence of 16:0/16:0-CPM-PA, the same PI molecular species as above were synthesized. However, with increasing concentrations of 16:0/16:0-CMP-PA in the medium, increasing amounts of labelled 16:0/16:0-PI appeared as well. With prepurified PI-synthase eluted from a DEAE column, endogenous CMP-PA was poorly utilized for PI biosynthesis whereas the exogenous 16:0/16:0-CPM-PA was used actively. With time, the endogenous CMP-PA was utilized first and the exogenous substrate was utilized, albeit, much more slowly. The results demonstrate that the selectivity displayed by PI-synthase towards various molecular species of CMP-PA depends on the integration of the enzyme in the membrane structure. Solubilization of the enzyme, i.e., inclusion of the protein in micelles with detergents and lipids, results in an apparent loss of the selectivity for CMP-PA.

Biosynthesis of gamma-linolenic acid in developing seeds of borage (Borago officinalis L.).

delta 6-desaturation of [14C]linoleoyl-CoA or [14C]oleoyl-CoA leading to the synthesis of gamma-linolenic acid was studied in vitro with microsomal fractions from developing seeds of Borago officinalis. Time course of the reaction, effects of protein and precursor concentrations and nucleotide requirements were examined. These parameters allowed us to improve the in vitro delta 6-desaturation assay. We observed that the precursors were acylated mainly in phosphatidylcholine, diacylglycerol and triacylglycerol, and then desaturated. NADH was absolutely required when [14C]oleoyl-CoA was the precursor, but not when [14C]linoleoyl-CoA was the precursor although it stimulated the reaction. The in vitro delta 6-desaturase activity was found mainly in phosphatidylcholine, associated with enriched endoplasmic reticulum membranes (ER) from embryos. No activity was observed in ER from seed coat or seedling. During maturation of the seeds, delta 6-desaturase reached its highest activity 14 to 16 days after pollination.

Biosynthesis of eukaryotic lipid molecular species by the cyanobacterium Spirulina platensis.

This report brings evidence that a prokaryotic photosynthetic organism can synthesize eukaryotic molecular species of glycerolipids. When Spirulina platensis PCC 8005 was supplemented with oleic acid, the sum of the percentages of 18 carbon (C18) fatty acids in monogalactosyldiacylglycerol (MGDG), the major lipid class, became largely higher than 50 mol%. This was absolutely unexpected from the well-known structure of cyanobacterial lipids. In these organisms, C18 fatty acids usually account for less than 50 mol% because they are esterified on carbon 1 of the glycerol, exclusively. This classical feature was 99% confirmed in control as well as in palmitate-supplemented cultures. The major molecular species of MGDG, which resulted from the different distributions of fatty acids on carbons 1 and 2 of glycerol, were C18/C16 type, belonging to the so-called "prokaryotic" type of lipids. By contrast, the molecular species of MGDG from oleate-supplemented cultures consisted of only 74 mol% of C18/C16 and the complementary 26 mol% were C18/C18, the so-called "eukaryotic" type of lipids. Furthermore, such C18/C18 lipids were also evidenced as traces (< 1%) in control cultures. These results underline the fact that the fatty acid specificity of 1-monoacylglycerol-3-phosphate-acyltransferase (in Spirulina) is not as absolute as the widely accepted concept of prokaryotic lipid would suggest. Oleate, supplemented at high concentration, can be compelled to act as a substrate for the acyltransferase and this results in the appearance of C18/C18 "eukaryotic" lipids in a prokaryotic organism.

In vitro photodynamic lipid peroxidation of total lipophilic extracts from leaves of bean plants.

In vitro lipid peroxidation, induced by reactive oxygen species photochemically generated, was carried out on purified alpha-linolenic acid (18:3(n - 3)) and on bean leaf total lipophilic extracts. The photosensitizer used was meso-tetraphenylporphine. The time-course of the reaction was evaluated by ultraviolet (UV) spectra analysis. The 18:3(n - 3) photoperoxidation kinetics comprised three steps: monohydroperoxidation, characterized by the appearance of conjugated diene patterns; dihydroperoxidation characterized by the appearance of conjugated triene patterns, oxidative cleavage of the mono- and dihydroperoxides, characterized by the disappearance of conjugated patterns. In contrast, for hydrated plant total lipophilic extract photoperoxidation, conjugated pattern appearance was slow and the maximum plateau was not obtained. In order to explain plant extract behaviour, we tested the effects of beta-carotene and quercetin (important components of the chloroplast membrane) on the time-course of 18:3(n - 3) photoperoxidation. The first step was inhibited by beta-carotene implying that type II photoperoxidation involving singlet oxygen (1O2) was predominant. Whilst the two last steps were inhibited by quercetin implying that type I photoperoxidation involving free radicals, prevailed. Since 18:3(n - 3) foliar content decreased under water deficit, we tested the behaviour of total lipophilic extracts from droughted plant leaves in presence of reactive oxygen species. In the case of droughted plants, the maximum of conjugated diene patterns was attained later than for hydrated plants, suggesting that drought affects the resistance capability of total lipophilic extracts from bean leaves to lipid photoperoxidation.

Comparison of the molecular species patterns of phosphatidic acid, CDP-diacylglycerols and phosphatidylinositol in potato tuber, pea leaf and soya-bean microsomes: consequences for the selectivity of the enzymes catalyzing phosphatidylinositol biosynthesis.

Microsomes prepared from pea leaf, potato tuber or germinated soya-beans, were incubated for 30 min with [14C]glycerol 3-phosphate. In the three tissues, phosphatidic acid (PA), CDP-diacylglycerols (CMP-PA) and phosphatidylinositol (PI) were labelled and could be separated by TLC. After methylation of phosphatidic acid, or treatment of CMP-PA by a nucleotidase, the molecular species composition of the three lipid classes could be determined by radio-HPLC. The similarity observed between the distributions of radioactivity among CMP-PA and PA molecular species, in the three tissues, indicates that the enzyme CTP:PA cytidylyltransferase did not present any selectivity towards any molecular species of PA. In contrast, only two molecular species containing palmitic acid (16:0/18:2 and 16:0/18:3) were labelled in PI whereas labelled PA and CMP-PA contained molecular species possessing stearic acid (18:0/18:2, 18:0/18:3 and 18:0/18:1). This indicates that the enzyme PI-synthase utilizes preferentially those molecular species of CMP-PA containing palmitic acid as substrates. However, mass analyses of PI prepared from the microsomes of the three tissues used in this study, indicated the presence of molecular species containing stearic acid (18:0/18:2 and 18:2/18:2). Except in soya-bean microsomes (where 18:0/18:2-PI represented 16% of total PI), those last molecular species were always present in small amounts.

Regulation by Lipids of Plant Microsomal Enzymes: III. Phospholipid Dependence of the Cytidine-Diphospho-Choline Phosphotransferase of Potato Microsomes.

Cytidine-diphospho-choline diacyl-glycerol phosphorylcholine phosphotransferase activity was demonstrated in potato (Solanum tuberosum L.) microsomes and the incorporation of cytidine-diphospho[(14)C]choline into phosphatidylcholine was characterized by the time course of (14)C incorporation and the effect of microsomal protein concentration on choline incorporation.Potato microsomes were progressively delipidated by treatments (2 min at 0 degrees C) with increasing amounts of phospholipase C from Bacillus cereus. A decrease in choline phosphotransferase activity was observed in parallel with the progressive hydrolysis of membrane phospholipids. A 70% (or more) phospholipid hydrolysis provoked the total inactivation of the enzyme.Adding back exogenous phospholipids (in the form of liposomes) to phospholipase C-treated membranes restored the enzymic activity. Restoration could be obtained with egg yolk phospholipids as well as with potato phospholipids. Restoration was time dependent and completed after 10 minutes; restoration was also dependent on the quantity of liposomes added to lipid-depleted membranes: the best restorations were obtained with 1 to 2.5 milligrams of phospholipid per mg of microsomal protein; higher phospholipid to protein ratios were less efficient or inhibitory.These results clearly demonstrate the phospholipid dependence of the cytidine-diphospho-choline phosphotransferase from potato microsomes.

Transfer of phospholipids between mesosomes and protoplasts from Bacillus subtilis.

1. Phospholipids were more intensively labelled from ammonium [1-14C]glycerophosphate in mesosomes than in protoplasts isolated from Bacillus subtilis. 2. When mesosomes, containing phospholipids labelled from sodium-[32P] phosphate were incubated with non radioactive protoplasts, labelled phospholipids were recovered in protoplasts after incubation. 3. This transfer of phospholipids from mesosomes toward protoplasts is time-dependent. 4. Soluble proteins obtained from Bacillus subtilis after ammonium sulphate precipitation were separated on a Sephadex G-100 column. 5. The two main fractions were able to accelerate the transfer of phospholipids from mesosomes toward protoplasts. 6. The first peak stimulated more actively the transfer of phosphatidylethanolamine whereas the second one preferentially accelerated the transfer of phosphatidylglycerol and diphosphatidylglycerol.

Regulation by Lipids of Plant Microsomal Enzymes: II. LIPID DEPENDENCE OF THE NADH-CYTOCHROME c REDUCTASE OF POTATO TUBERS.

Microsomal membranes from potato tubers were treated with a phospholipase C extracted from Bacillus cereus. A positive correlation could be observed between the hydrolysis of membranous phospholipids and the decrease of the NADH-cytochrome c reductase activity. Addition of total lipid or phospholipid micelles to phospholipase C-treated microsomes partially restored the NADH-cytochrome c reductase activity, thus proving the lipid-dependence of this enzyme.

Hepatic delta 9 and delta 6 desaturase activities during the recovery period following carbon tetrachloride poisoning.

The liver microsomal delta 9 and delta 6 desaturase activities have been studied in rats with carbon tetrachloride-induced hepatitis. Immediately after poisoning, significant decreases were observed for both types of desaturase activity. However, recovery kinetics were slower for the delta 6 desaturase than for the delta 9 desaturase. The activities of NADH-ferricyanide and NADH-cytochrome C reductases, proteins involved in the electron transfers associated with microsomal desaturation, were also measured. There was a fall in both activities after poisoning, but this decrease was less than that of the desaturase activities.

[Formation of polyunsaturated fatty acids in plants]. / Formation des acides gras polyinsaturés dans le régne végétal.

Higher plants synthesize large amounts of linoleic and alpha-linolenic acids by progressive desaturation of oleic acid. Plant desaturases are active only during a short period of the development of seeds or leaves. 14C-oleate desaturation has been followed experimentally in various plants (Rapeseed, Sunflower, Flax and Potato). With aged potato slices, it has been shown that oleate-desaturase is an inducible enzymatic system. With microsomes isolated from aged potato-slices, 14C-oleoyl-CoA has been desaturated in vitro. Recent results suggest that oleoyl-phosphatidylcholine is the true substrate of the desaturation system. The formation of the alpha-linolenic acid of chloroplasts has not been obtained in vitro, up to now. In vivo, the newly formed linolenic acid is found in chloroplast galactolipids. In conclusion, difference between animal and plant desaturases are underlined.

Hepatic Δ(9) and Δ(6) desaturase activities during the recovery period following carbon tetrachloride poisoningdesaturase activities during the recovery period following carbon tetrachloride poisoning.

The liver microsomal Δ(9) and Δ(6) desaturase activities have been studied in rats with carbon tetrachloride-induced hepatitis. Immediately after poisoning, significant decreases were observed for both types of desaturase activity. However, recovery kinetics were slower for the Δ(6) desaturase than for the Δ(9) desaturase. The activities of NADH-ferricyanide and NADH-cytochrome C reductases, proteins involved in the electron transfers associated with microsomal desaturation, were also measured. There was a fall in both activities after poisoning, but this decrease was less than that of the desaturase activities.

[Effect of increasing concentrations of detergents on electron transfer in chloroplasts]. / Effets de concentrations croissantes de détergents sur les transferts d'électrons dans les chloroplastes.

The effects of increasing concentrations of sodium cholate and deoxycholate, "Tween 80" and "Triton X 100" upon O2 evolution by intact or osmotically broken spinach chloroplasts were investigated polarographically. Except for cholate, low concentrations of any detergent increased considerably (up to four times) the rates of oxygen evolution by broken chloroplasts, without interrupting the electron flux between photosystem II and photosystem I.

[Role of lipids in the function of microsomal electron transport chains of potato]. / Rôle des lipides dans le fonctionnement des chaînes de transport d'électrons de microsomes de Pomme de terre.

Microsomal membranes from potato tubers were extracted by acetone solutions of increasing concentrations (5, 10, 15, 20, 30, 40, 50, 70 and 90 p. cent). Microsomal lipids were progressively extracted: acetone concentrations exceeding 30 p. cent extracted large amounts of membraneous phospholipids (figure). Lipid extraction reduced NADH-cytochrome c reductase activity but did not affect NADH-ferricyanide reductase and NADPH-cytochrome c reductase activities. This was confirmed by experiments using increasing concentrations of sodium deoxycholate. After lipid extraction with acetone (or solubilization by triton X100), NADH-cytochrome c reductase activity of microsomal membranes could not be recovered by adding back lipids under various experimental conditions. These results strongly suggest that, in potato microsomes, lipids are undispensable components of the electron transport chain starting from NADH especially in the portion involving cytochrome b5. On the contrary, the second microsomal electron transport chain, starting from NADPH, is not regulated by lipids. However, plant microsomal membranes would be much more disturbed by liped extraction than animal microsomes and suitable relipidation conditions remain to be found to prove definitely the lipid dependence of plant microsomal electron transport.

[Membrane lipids and electron transfer. Effects of four detergents on NADH-ferricyanide reductase and NADH-cytochrome c reductase activities of potato tuber microsomes]. / Lipides membranaires et transferts d'électrons. Effets de quatre détergents sur les activitiés NADH-ferricyanure réductase et NADH-cytochrome c réductase de microsomes isolés du tubercule de Pomme de terre.

The NADH-ferricyanure reductase activity of Potato microsomes is stimulated by non ionic detergents (Triton X100 and Tween80) and is partially inhibited by ionic detergents (sodium-cholate and deoxycholate). All these four detergents progressively decreased the NADH-cytochrome c reductase in the following order: sodium deoxycholate greater than Triton X100 greater than sodium cholate greater than Tween80.

[The effects of 4 detergents on the respiration of mitochondria isolated from rat liver]. / Effets de quatre détergents sur la respiration de mitochondries isolées du foie de rat

The effects of these four detergents (two non-ionic: Titron X100 and Tween 80, two ionic: sodium cholate and sodium dedeoxycholate) upon the respiratory intensities of mitochondria and upon the ADP/0 and respiratory control ratios were observed. At low concentrations and in the absence of exogenous ADP, non ionic as well as ionic detergents provoked a threefold (or fourfold) increase of the respiratory intensities of mitochondria. At higher concentrations, the four detergents were inhibitory for mitochondrial oxidations in the order: Triton X 100 greater than DOC greater than cholate greater than Tween 80. At increasing doses, the four detergents progressively decreased the phosphorylating capacities of mitochondria.