Biosynthesis of monogalactosyl diglyceride by chloroplasts from spinacia oleracea and from some gramineae

Synthetic diglycerides which differed in unsaturation of fatty acids gave the same incorporation of [¹⁴C]galactose from UDP-[¹⁴C]galactose when added to acetone powders of spinach chloroplasts up to about 0·6 mg diglyceride/20 mg acetone powder. Diolein and the endogenous diglyceride isolated from the acetone extract of chloroplasts stimulated galactolipid biosynthesis to a similar extent. With all diglycerides used, monogalactosyl diglyceride was the main product with little accompanying synthesis of digalactosyl diglyceride. The radioactivity in the monogalactosyl diglyceride synthesized from UDP-[¹⁴C]galactose by whole chloroplasts was distributed widely among the monogalactosyl diglycerides with different fatty acid composition. It is concluded that the enzyme which catalyses the transfer of galactose from UDP-galactose to diglyceride is not specific for polyunsaturated diglycerides and that the polyunsaturated monogalactosyl diglycerides arise either by desaturation of the fatty acyl residues after monogalactolipid synthesis or by transacylation. Acetone powders of chloroplasts prepared from several Gramineae did not exhibit transferase activity although whole chloroplasts were active.     

Structural studies on the diglyceride-mediated activation of protein kinase C

Diglyceride analogs were studied with respect to their abilities to activate protein kinase C (Ca2+- and phospholipid-dependent protein kinase) in the presence of low calcium and phospholipid. Analogs which lacked either a free hydroxyl group at the 3 position or an ester moiety at the 1 position were without activity. It was concluded that the hydrophilic moieties of the active diglycerides are crucial for activity. However, diglyceride analogs containing additional hydrophilic moieties in one of the acyl side chains did not exhibit enhanced activity when compared to diglycerides containing two fatty acyl groups. Diglyceride analogs with a modified glycerol backbone were also studied. Homologous diglycerides with either one or two methylene groups between the 3-methylene group of the diglyceride and the hydroxyl group possessed markedly reduced activities when compared to the appropriate unmodified diglyceride. Isomers of these homologues which contained either a methyl group at the 1 position, or dimethyl groups incorporated at the 1 and 3 positions, were virtually without activity. Where studied, none of the diglyceride analogs prepared possessed antagonist activity. The results of these experiments are discussed with respect to the extreme specificity observed.     

Lipid composition of Mycoplasma neurolyticum

The total lipid content of Mycoplasma neurolyticum comprises about 14% of the dry weight of the organisms and is about equally distributed between the phospholipid and the neutral-glycolipid fractions. The neutral lipids were identified as triglycerides, diglycerides, and cholesterol. The glycolipid fraction contained 1-O-beta-glucopyranosyl-d-2,3-diglyceride and 1-[O-beta-d-glycopyranosyl-(1-->6)-O-beta-d-glucopyranosyl]-d-2,3-diglyceride. The latter lipid is structurally identical to the diglucosyl diglyceride which occurs in Staphylococcus aureus. The phospholipids of the organism consist of a fully acylated glycerophosphoryl-glycerophosphoryl glycerol, phosphatidic acid, diphosphatidyl glycerol, phosphatidyl glycerol, and amino acyl esters of phosphatidyl glycerol. Phosphatidic acid and phosphatidyl glycerol account for greater than 90% of the phospholipids of organisms in the exponential phase of growth. The predominant fatty acids found in all of the acyl lipids were palmitic, stearic, and oleic acids.     

Degradation of monogalactosyl diglyceride and diagalactosyl diglyceride by sheep pancreatic enzymes

1. Saline extract of sheep pancreas acetone-dried powder was shown to catalyse acyl ester hydrolysis of spinach leaf galactosyl diglycerides and also galactosylglucosyl diglyceride of Lactobacillus casei. 2. Sodium deoxycholate stimulated the enzyme activity. Ca(2+) had no effect on the hydrolysis of monogalactosyl diglyceride, but it enhanced that of digalactosyl diglyceride. When added together, there was considerably less activity with both the substrates. 3. Optimal hydrolysis was observed at pH7.2. 4. The initial point of hydrolysis was at position-1, leading to the formation of monogalactosyl monoglyceride and digalactosyl monoglyceride. Further hydrolysis to the corresponding galactosylglycerols and later to galactose and glycerol was also observed, indicating the presence of alpha- and beta-galactosidases in the enzyme preparation. 5. Formation of monogalactosyl diglyceride from digalactosyl diglyceride by the action of alpha-galactosidase was noted. 6. Monogalactosyl diglyceride was also hydrolysed by beta-galactosidase to a limited extent, giving rise to diacylglycerol and galactose. 7. Attempts at purification of monogalactosyl diglyceride acyl hydrolase by using protamine sulphate treatment, Sephadex G-100 filtration and DEAE-cellulose chromatography gave a partially purified enzyme which showed 9- and 81-fold higher specific activity towards monogalactosyl diglyceride and digalactosyl diglyceride respectively. This still showed acyl ester hydrolysis activity towards methyl oleate, phosphatidylcholine and triacylglycerol. 8. When sheep, rat and guinea-pig tissues were compared, guinea-pig tissues showed the highest activity towards both monogalactosyl diglyceride and digalactosyl diglyceride. In all the species pancreas showed higher activity than intestine.     

Biosynthesis of Triglyceride and Other Fatty Acyl Esters by Developing Rat Brain

Abstract: The biosynthesis of triglyceride from 1,2-diglyceride and long-chain acyl coenzyme A (CoA) was studied in developing rat brain. Diglyceride acyltransferase activity was highest in a microsomal fraction, had a neutral pH optimum, and was stimulated by MgCl₂. Palmitoyl CoA and oleoyl CoA served equally well as acyl donors. The enzyme catalyzed the acylation of both endogenous diglyceride and several naturally occurring and synthetic exogenous diglycerides. In addition, short-chain primary and secondary alcohols were found to be acylated under these conditions. A second acylation system, active at low pH, was found to catalyze esterification of ethanol and cholesterol, but not diglyceride, with free fatty acid. These results demonstrate that brain has the capacity to acylate a wide variety of physiological and nonphysiological hydroxyl compounds.     

Influence of S-ethyl Dipropylthiocarbamate (EPTC) on the Fatty Acid Composition of Wheat Leaf Galactolipids

Winter wheat (Triticum sativum L. cv. Nisu) was grown in sand which contained 0, 0.25, 0.5 and 1.0 mg S-ethyl dipropylthiocarbamate (EPTC) per kg air dry sand. The galactolipids of leaves of 21-day-old seedlings were isolated by preparative thin layer chromatography. The fatty acids of the mono- and digalactosyl diglycerides were analysed by gas liquid capillary chromatography. The major fatty acids of the wheat leaf galactolipids were palmitic, palmitoleic, stearic, oleic, vaccenic, linoleic and linolenic acids (in the monogalactosyl diglyceride fraction of untreated plants 22.5, 2.4, 3.1, 5.2, 2.5, 51.1 and 1526.6 μg and in the digalactosyl diglyceride fraction 108.8, 2.3, 10.4, 9.9, 8.2, 42.3 and 1120.7 μg/g leaf fresh weight, respectively). Total fatty acid content of the mono- and digalactosyl diglyceride fractions was decreased by 85 and 87%, respectively, at 1 mg EPTC/kg sand, while decrease in the fresh weight of the leaves was 79%. The content of linoleic and linolenic acids/g fresh weight of the leaves was decreased in the monogalactosyl diglyceride fraction by 27 and 43%, respectively, while the content of all other fatty acids was increased. In the digalactosyl diglyceride fraction the content of both linoleic and linolenic acids/g leaf fresh weight was decreased by 55%. The content of palmitic and vaccenic acids was also decreased, whereas the content of other fatty acids remained at the level of the untreated samples. The general quality of the fatty acids in the mono- and digalactosyl diglyceride fractions was altered slightly by EPTC.     

Lipase-catalyzed ester exchange reactions in organic media with controlled humidity

Immobilized lipase activity is studied in organic solvent systems of controlled water content under the influence of a variety of reaction parameters, such as temperature, relative humidity, substrate concentrations, and type of fatty acid used. Control of the amount of water in the reaction system was found to be a valuable tool for the orientation of the reaction process and for the determination of the final reaction products. The properties of the immobilized lipase were studied using the interesterification of triolein and palmitic acid as the model system.     

Lipids in Cruciferae IX. The Effect of Growth Temperature and Stage of Development on the Fatty Acid Composition of Leaves, Siliques and Seeds of "Zero-erucic-acid" Breeding Lines of Brassica napus

Two breeding lines of “zero-erucic-acid” rapeseed (Brassica napus) were grown in climate chambers at a constant night temperature (12°C) and constant photoperiod (16 hours) but with different day temperatures (15, 20 and 25°C). Samples of leaves, siliques and immature seeds were analysed for total fatty acid pattern. The content of different acyl lipids and the fatty acid pattern of these lipids were also determined in some of the samples by use of preparative TLC followed by GLC of the fatty acids. The mature seeds produced by ten plants of each selection in each climate were analysed separately for total fatty acid composition. Mono- and digalactosyl diglycerides (MGDG, DGDG) were the predominant acyl lipids in leaves and siliques. In developing seeds they also were more abundant than the phospholipids, but in this case the neutral lipids, mainly triacylglycerols, contained about 95% of the total fatty acids. Large variations were found in the fatty acid composition of monogalactosyl diglyceride and digalactosyl diglyceride, isolated from leaves, siliques and immature seeds. The palmitic acid content of leaf MGDG was about 15 %, atypically high for MGDG from photosynthetic tissue. The linolenic acid content of the MGDG was about 45 %, 30 % and 10 % in the leaf, silique and seed tissues respectively. A hexadecatrienoic acid (16: 3) was found almost exclusively in the MGDG samples of leaves, siliques and immature seeds (about 25 %, 10 % and 3 % 16:3 respectively). The lipids of siliques — mainly photosynthetising tissue — were different from those of leaves and had especially high contents of stearic acid (6–12 % in the different lipids). For all lipid classes studied, leaves grown at the lowest day temperature had a slightly lower oleic and higher linolenic acid content than those grown at the highest temperature. On the other hand, increasing the day temperature caused a decreased level of oleic, an increased level of linoleic and an essentially unchanged level of linolenic acids in the mature seeds from both selections.     

Obtaining monoglycerides by esterification of glycerol with palmitic acid using some high activity preparations of Candida antarctica lipase B

Cross-linked enzyme aggregates (CLEAs), protein coated microcrystals (PCMCs), cross-linked protein coated microcrystals (CLPCMCs) of Candida antarctica lipase B (CALB) were used for esterification of glycerol with palmitic acid in acetone under low water condition. With CLEAs, 81% monoglyceride (MG) along with 4.5% diglyceride (DG) were produced at 1% (v/v) water content in 24 h. The water content in the medium was managed by stepwise addition of the molecular sieves at appropriate time intervals. With PCMCs (potassium sulfate as a core material), 82% monoglyceride along with 4.0% diglyceride were obtained, with 0.5% water (v/v) added initially to anhydrous acetone with molecular sieves present in the reaction medium. With CLPCMC (prepared by cross-linking with 200 mM glutaraldehyde), 87% monoglyceride and 3.3% diglyceride were produced in 24 h in presence of 1% (v/v) water (added initially) and with appropriate amount of molecular sieves added in the reaction medium. The results offer a comparative study on the performance of three high activity preparations of CALB for preparation of monopalmitin with ≤10% of the diglyceride content.     

Quantitative and compositional changes in monogalactosyl and digalactosyl diglycerides during light-induced formation of chloroplasts in Euglena gracilis

The formation of chloroplasts in dark-grown cells of Euglena gracilis was induced by exposing the cells to constant illumination. Following a lag, the cells accumulated chlorophyll and galactosyl diglycerides simultaneously at almost linear rates. The monogalactosyl diglyceride content rose from approximately 2 micromoles in 100 mg of dark-grown cells to 27 micromoles in fully green cells; the digalactosyl diglyceride content increased from 1 micromole to 11 micromoles. The digalacto compounds increased more rapidly than the monogalacto compounds at first, but their rate of accumulation began to diminish long before greening of the cell was complete. The sole exception was the digalactosyl diglyceride fraction that contained hexadecadienoic (16:2) fatty acid. This fraction increased continuously during greening. As accumulation of the digalacto compounds diminished, that of the monogalacto compounds increased. Towards the end of greening, the major fatty acids were 16:2, 16:3, 16:4, 18:2, and 18:3 in the monogalacto and 16:2 in the digalacto compounds. The results of this study suggest that monogalactosyl and digalactosyl diglycerides that contain particular fatty acid components have a function in the assembly of chloroplasts.     

Wax esters synthesis from heavy fraction of sheep milk fat and cetyl alcohol by immobilised lipases

Wax esters were obtained from lipase-catalysed alcoholysis of triglycerides with cetyl alcohol, using n-hexane as solvent. The heavy triglyceride fraction (HTF), obtained by fractionation of sheep milk fat, was used as raw material. In the natural fat mixture GC analysis showed that palmitic, myristic, stearic and oleic acids are the most abundant fatty acids which are useful to produce wax esters. Reactions were tested for different amounts of Lipozyme RMIM catalyst, and the optimum concentration of 10 mg catalyst/ml solution has been determined. The formation of the four main products, i.e. cetyl myristate, cetyl palmitate, cetyl oleate and cetyl stearate, was determined by HPLC/ELSD quantitative analysis. The optimum water activity in the reaction medium a_w=0.35 in the case of Lipozyme RMIM, and a_w=0.53 for Novozym 435 was found. Lipozyme RMIM (immobilised sn-1,3-specific lipase from Rhizomucor miehei) was more active than Novozym 435 (immobilised nonspecific lipase-B from Candida antarctica) towards wax esters production. The acyl migration of 2-monoglycerides was suggested as a crucial step to explain the higher yields produced by the 1,3-specific lipase.     

Antiviral nucleoside diphosphate diglycerides: improved synthesis and facilitated purification.

Cytidine diphosphate diglyceride and its analogs have previously been synthesized by condensing phosphatidic acid with the monophosphomorpholidates of the various nucleosides. Yields have been low and purification of the product has been difficult. We report here an improved method for the synthesis of nucleoside diphosphate diglycerides with potential antiviral activity. Phosphatidic acid was activated with morpholine in the presence of dicyclohexylcarbodiimide to phosphatidic acid morpholidate. This compound was condensed with the 5'-monophosphate of the anti-HIV agents 3'-azido-3'-deoxythymidine, 3'-deoxythymidine or 2',3'-dideoxycytidine, and the monophosphate of the anti-HSV agent acyclovir. The resulting nucleoside diphosphate diglycerides are potential candidates for improved antiviral action when compared to the parent nucleoside analogs. Compared to the older method for the preparation of cytidine diphosphate diglyceride and analogs thereof, the new method has several advantages: reaction times are reduced from several days to several hours and the yield of the reactions is generally increased from 20-40% to between 50 and 80%. In addition, the purification of the compounds is greatly facilitated due to the small amount of phosphatidic acid remaining in the reaction mixture.     

Biosynthesis of galactolipids by enzyme preparations from spinach leaves

The pH optimum for galactolipid synthesis from UDP-galactose by spinach chloroplasts is 7.2 in Tris-HCl or phosphate buffer. The products include sterol glycosides, trigalactosyl diglyceride (tentatively identified), digalactosyl diglyceride, and monogalactosyl diglyceride in increasing order of quantity. The proportion of monogalactosyl diglyceride decreases and that of digalactosyl diglyceride increases as the pH is lowered. The galactolipid synthesis is quite resistant to elevated temperature; maximal incorporation of galactose from UDP-galactose was observed at 45 degrees C. The proportion of monogalactosyl diglyceride was greater at the higher temperatures. As much as 40% of the galactolipid-synthesizing capability of a spinach leaf homogenate is not sedimented by centrifugation for 60 min at 100,000 g. An acetone powder of spinach chloroplasts contains enzymes which catalyze galactolipid synthesis. This preparation is dependent on added diglycerides in order to make galactolipid, whereas the chloroplast preparation is not dependent on added diglycerides. Molecular species of diglycerides were compared as requirements for galactolipid synthesis. The requirement was satisfied best by the diglycerides of highest unsaturation. Methylation of the free hydroxyl of the diglyceride eliminated the effectiveness.     

Enzymatic fatty acid exchange in digalactosyldiacylglycerol

Six different lipases were screened for their ability of acidolysis between digalactosyldiacylglycerol (DGDG) and heptadecanoic acid in toluene. Lipases from Geotrichum candidum, Alcaligenes sp. and Penicillium camembertii did not catalyse the acidolysis reaction. Rhizopus arrhizus and Rhizomucor miehei (Lipozyme) catalysed the acidolysis but produced a mixture of DGMG, DGDG, acyl-DGMG and acyl-DGDG. The extra acyl group is bound to the primary hydroxyl of the digalactosyl moiety. Candida antarctica also catalysed the acidolysis but the TLC analysis showed bands with higher Rf values than acyl-DGDG, these probably being different tetra and higher esters. R. arrhizus lipase was the most promising enzyme under the conditions used, with no tetra esters being formed and giving the highest reaction rate of the enzymes investigated. Low water activity (0.06 or 0.11) and high fatty acid concentration (400 mM) increased the formation of acyl-DGDG whilst higher water activities (0.33 and 0.54) increased the amount of DGMG when R. arrhizus lipase was used as catalyst. At a water activity of 0.11 and a fatty acid concentration of 400 mM a yield of 24% modified DGDG was obtained. In this product the fatty acid originally present in the sn-1 position had been exchanged by heptadecanoic acid.     

Four-directional-development thin-layer chromatography of lipids using trimethyl borate

Solvent mixtures containing trimethyl borate virtually eliminated the pronounced interconversion of 1,2- and 1,3-dipalmitins during their resolution by thin-layer chromatography on Silica Gel G. With trimethyl borate, an average of 1-2% of 1,2-dipalmitin was converted to 1,3-dipalmitin. A four-directional-development TLC procedure incorporating trimethyl borate resolves cholesteryl glucoside, ceramides, monogalactosyl diglyceride, 1- and 2-monopalmitin, palmitic acid, cholesterol, 1,2- and 1,3-dipalmitin, tripalmitin, methyl palmitate, cholesteryl palmitate, beta-carotene and some of its degradation products, squalene, and tetracosane. Digalactosyl diglyceride, phosphatidic acid, phosphatidylglucose, cerebrosides, and other phospholipids remain near the origin. A mixture containing triolein, 1,2- and 1,3-diolein, 1- and 2- monoolein, oleic acid, and cholesterol was resolved in one dimension. A similar series of palmitic-containing neutral lipids was also resolvable in one dimension. These procedures were applied to the TLC of human sera lipids.     

Structural analysis of phosphatidylcholine of plant tissue

Pure preparations of phosphatidylcholine were isolated from spinach leaf chloroplasts, spinach leaf microsomes, and cauliflower inflorescence. The isolated phosphatidylcholine was treated with snake venom phospholipase A, and the fatty acid distribution and composition of the fatty acid methyl esters prepared from the lysophosphatidylcholine and the freed fatty acid were determined by gas-liquid chromatography. The results showed that saturated fatty acids were preferentially esterified at position 1 and unsaturated fatty acids at position 2. The phosphatidylcholine from cauliflower was also treated with phospholipase C. The resulting diglycerides were fractionated on AgNO(3)-impregnated thin-layer plates. The diglyceride fractions were transesterified and the fatty acid composition of each was determined by gas-liquid chromatography. The predominant species contained linolenic acid only (22% of the total), linolenic and oleic acids (19%), and linolenic and palmitic acids (37%). These molecular species could not be accounted for by random distribution of the fatty acids.     

Lipase-catalyzed synthesis of oleic acid esters of polyethylene glycol 400

Summary Quantitative esterification of polyethylene glycol (PEG) 400 using oleic acid and Lipozyme was achieved in hexane. The effects of temperature, nature of acyl donor, substrate ratio, enzyme quantity and reaction time upon PEG esterification were examined. Nearly selective production of either PEG monooleate or PEG dioleate was achieved. Lipozyme was still 80% active after five reaction cycles.     

Biosynthesis of mitochondrial phospholipids using endogenously generated diglycerides.

When isolated mitochondria or microsomes from rat liver were treated with phospholipase C, the incorporation of radioactive phospholipid precursors was markedly enhanced, presumably as a result of production of diglycerides by hydrolysis of endogenous phospholipids. Incorporation of CDP[14C]choline into lecithin in rat liver or BHK-21 mitochondria could be attributed to residual contamination from elements of the endoplasmic reticulum, with added diglycerides or with endogenous diglycerides produced by the phospholipase C treatment. A similar stimulation of [gamma32P]ATP incorporation into phospholipids was observed with exogenous or endogenous diglycerides, but the mitochondrial diglyceride kinase in either case was also related to the degree of microsomal contaminants. It was concluded that previous studies showing negligible capacity of mitochondria for lecithin biosynthesis de novo were not explainable on the basis of limited accessibility of added diglycerides, and that formation of phosphatidic acid by diglyceride kinase was not of significance in rat liver mitochondria.     

Substrate Specificity and Mode of Action of the Lipase of Thermophilic Fungus Humicola lanuginosa S-38

Substrate specificity of the lipase of thermophilic fungus, Humicola lanuginosa S–38, was investigated. It was found that the lipolytic activity was greatly influenced by the structure of both fatty acid and alcohol moieties of the substrate. It was concluded that the hydrolysis of both water soluble and water insoluble ester was catalyzed by the Humicola lipase itself. The Humicola lipase showed no positional specificity and split ester bonds on all positions of triolein at about the same rate. Both palmitic acid (α) and linoleic acid (β) ester bonds of phosphatidyl-ethanolamine were split indicating no positional specificity of fatty acid ester bonds. From above results, it was made clear that mode of action of Humicola lipase on triolein and on phosphatidyl-ethanolamine is identical. The Humicola lipase had no activity of lipoprotein lipase.     

Source and role of diacylglycerol formed during phagocytosis of opsonized yeast particles and associated respiratory burst in human neutrophils

The results presented in this paper demonstrate that in human neutrophils phagocytosis of C3b/bi and IgG-opsonized yeast particles is associated with activation of phospholipase D and that this reaction is the main source of diglycerides. The demonstration is based upon the following findings: 1) the challenge of neutrophils with these opsonized particles was followed by a rapid formation of [3H]alkyl-phosphatidic acid [( 3H]alkyl-PA) and [3H]alkyl-diglyceride [( 3H]alkyl-DG) in cells labeled with [3H]alkyl-lyso-phosphatidylcholine; 2) in the presence of ethanol [3H]alkyl-phosphatidylethanol was formed, and accumulation of [3H]alkyl-PA and [3H]alkyl-DG was depressed; 3) propranolol, by inhibiting the dephosphorylation of [3H]alkyl-PA, completely inhibited the accumulation of [3H]alkyl-DG and depressed by about 75% the formation of diglyceride mass. Evidence is also presented that phagocytosis of C3b/bi and IgG-opsonized yeast particles and associated respiratory burst can take place independently of diglyceride formation and of the activity of this second messenger on protein kinase C. In fact: a) propranolol while completely inhibited the formation of diglyceride mass did not modify either the phagocytosis or respiratory burst; b) these two processes were insensitive to staurosporine.