Semi-synthetic approach for the preparation of homogeneous plasmenylethanolamine utilizing phospholipase D from Streptomyces chromofuscus

A simple method for the preparation of homogeneous molecular species of plasmenylcholine and plasmenylethanolamine was developed. The method utilized reverse phase high performance liquid chromatography to isolate homogeneous molecular species of plasmenylcholine prepared by acylation of lysoplasmenylcholine. Plasmenylcholine was directly converted to plasmenylethanolamine by transphosphatidylation utilizing phospholipase D from Streptomyces chromofuscus. This method permits the facile labeling of homogeneous molecular species of plasmalogens in the polar head group, the sn-2 acyl chain, or both, for the first time.     

Phospholipid spin probes measure the effects of ethanol on the molecular order of liver microsomes

Ethanol, in vitro, is known to perturb the molecular order of the phospholipids in biological membranes, while chronic ethanol exposure, in vivo, leads to resistance to disordering. Such changes have usually been measured by electron spin resonance, utilizing fatty acid spin probes. The use of such probes is controversial, since their orientation in the membrane may not accurately represent that of individual phospholipids. We, therefore, compared ethanol-induced structural perturbations in the membranes of rat hepatic microsomes measured with the spin probe 12-doxylstearic acid (SA 12) with those assayed with various phospholipid spin probes. With SA 12, the addition of increasing amounts of ethanol (50-250 mM) in vitro caused a progressive decrease in the membrane molecular order, as measured by electron spin resonance (ESR). By contrast, microsomes obtained from rats chronically fed ethanol were resistant to the disordering effect of ethanol. Microsomes labeled with the phospholipid spin probes 1-palmitoyl-2-(12-doxylstearoyl)phosphatidylcholine, -phosphatidylethanolamine, or -phosphatidic acid also exhibited increased disordering with the addition of increasing amounts of ethanol. However, the effect noted with phospholipid spin probes was less than that observed with the fatty acid probe. Microsomes obtained from the livers of chronically intoxicated animals labeled with the phospholipid probes were also resistant to the disordering effects of ethanol in vitro. These results suggest that fatty acid spin probes are qualitatively valid for measuring membrane perturbations in biological membranes, ethanol affects all microsomal phospholipids, regardless of chemical dissimilarities (e.g., head-group structure), in a qualitatively similar fashion, and the fluidization of fatty acyl chains in microsomal membranes is comparable in different membrane phospholipids.     

Chain-Length Heterogeneity Allows for the Assembly of Fatty Acid Vesicles in Dilute Solutions

A requirement for concentrated and chemically homogeneous pools of molecular building blocks would severely restrict plausible scenarios for the origin of life. In the case of membrane self-assembly, models of prebiotic lipid synthesis yield primarily short, single-chain amphiphiles that can form bilayer vesicles only at very high concentrations. These high critical aggregation concentrations (cacs) pose significant obstacles for the self-assembly of single-chain lipid membranes. Here, we examine membrane self-assembly in mixtures of fatty acids with varying chain lengths, an expected feature of any abiotic lipid synthesis. We derive theoretical predictions for the cac of mixtures by adapting thermodynamic models developed for the analogous phenomenon of mixed micelle self-assembly. We then use several complementary methods to characterize aggregation experimentally, and find cac values in close agreement with our theoretical predictions. These measurements establish that the cac of fatty acid mixtures is dramatically lowered by minor fractions of long-chain species, thereby providing a plausible route for protocell membrane assembly. Using an NMR-based approach to monitor aggregation of isotopically labeled samples, we demonstrate the incorporation of individual components into mixed vesicles. These experiments suggest that vesicles assembled in dilute, mixed solutions are depleted of the shorter-chain-length lipid species, a finding that carries implications for the composition of primitive cell membranes.     

Chain-Length Heterogeneity Allows for the Assembly of Fatty Acid Vesicles in Dilute Solutions

A requirement for concentrated and chemically homogeneous pools of molecular building blocks would severely restrict plausible scenarios for the origin of life. In the case of membrane self-assembly, models of prebiotic lipid synthesis yield primarily short, single-chain amphiphiles that can form bilayer vesicles only at very high concentrations. These high critical aggregation concentrations (cacs) pose significant obstacles for the self-assembly of single-chain lipid membranes. Here, we examine membrane self-assembly in mixtures of fatty acids with varying chain lengths, an expected feature of any abiotic lipid synthesis. We derive theoretical predictions for the cac of mixtures by adapting thermodynamic models developed for the analogous phenomenon of mixed micelle self-assembly. We then use several complementary methods to characterize aggregation experimentally, and find cac values in close agreement with our theoretical predictions. These measurements establish that the cac of fatty acid mixtures is dramatically lowered by minor fractions of long-chain species, thereby providing a plausible route for protocell membrane assembly. Using an NMR-based approach to monitor aggregation of isotopically labeled samples, we demonstrate the incorporation of individual components into mixed vesicles. These experiments suggest that vesicles assembled in dilute, mixed solutions are depleted of the shorter-chain-length lipid species, a finding that carries implications for the composition of primitive cell membranes.     

The primary determinant of rabbit myocardial ethanolamine phosphotransferase substrate selectivity is the covalent nature of the sn-1 aliphatic group of diradyl glycerol acceptors.

Plasmenylethanolamines represent the major endogenous phospholipid storage depot of arachidonic acid in many mammalian cells. To elucidate the biochemical mechanisms contributing to the high plasmalogen content and arachidonic acid enrichment present in myocardial ethanolamine glycerophospholipids, the substrate specificity of rabbit myocardial ethanolamine phosphotransferase (EPT) was quantified utilizing multiple molecular species of each subclass of diradyl glycerol substrate. Myocardial EPT demonstrated over a 16-fold selectivity for 1-O-alk-1'-enyl-2-acyl-sn-glycerol (AAG) compared to 1,2-diacyl-sn-glycerol (DAG) substrate utilizing individual molecular species of each subclass dispersed in Tween 20. The selective utilization of AAG by EPT was substantiated utilizing two independent assay systems which employed either the presentation of substrate to enzyme as a substitutional impurity in Triton X-100 mixed micelles or the obligatory utilization of endogenously generated diradyl glycerol substrates. Although rabbit myocardial microsomes contained over a 20-fold molar excess of endogenous DAG to AAG mass, incubation of rabbit myocardial microsomes with CDP-ethanolamine resulted in the highly selective synthesis of plasmenylethanolamines which were predominantly comprised of molecular species containing arachidonic acid at the sn-2 position (greater than 75%). Endogenous AAG molecular species in rabbit myocardial microsomes were similarly enriched in arachidonic acid, and the distribution of AAG molecular species closely paralleled the distribution of plasmenylethanolamine (but not plasmenylcholine) molecular species. Thus, the subclass and molecular species distribution of the ethanolamine glycerophospholipids synthesized by rabbit myocardial EPT reflects independent contributions from the subclass selectivity of EPT for AAG substrate in conjunction with the enrichment of arachidonic acid in microsomal AAG molecular species.     

Disparate molecular dynamics of plasmenylcholine and phosphatidylcholine bilayers

The molecular dynamics of binary dispersions of plasmenylcholine/cholesterol and phosphatidylcholine/cholesterol were quantified by electron spin resonance (ESR) and deuterium magnetic resonance (2H NMR) spectroscopy. The order parameter of both 5-doxylstearate (5DS) and 16-doxylstearate (16DS) was larger in vesicles comprised of plasmenylcholine in comparison to phosphatidylcholine at all temperatures studied (e.g., S = 0.592 vs. 0.487 for 5DS and 0.107 vs. 0.099 for 16DS, respectively, at 38 degrees C). Similarly, the order parameter of plasmenylcholine vesicles was larger than that of phosphatidylcholine vesicles utilizing either spin-labeled phosphatidylcholine or spin-labeled plasmenylcholine as probes of molecular motion. The ratio of the low-field to the midfield peak height in ESR spectra of 16-doxylstearate containing moieties (i.e., spin-labeled plasmenylcholine and phosphatidylcholine) was lower in plasmenylcholine vesicles (0.93 +/- 0.01) in comparison to phosphatidylcholine vesicles (1.03 +/- 0.01). 2H NMR spectroscopy demonstrated that the order parameter of plasmenylcholine was greater than that of phosphatidylcholine for one of the two diastereotopic deuterons located at the C-2 carbon of the sn-2 fatty acyl chain. The spin-lattice relaxation times for deuterated plasmenylcholine and phosphatidylcholine in binary mixtures containing 0-50 mol % cholesterol varied nonmonotonically as a function of cholesterol concentration and were different for each phospholipid subclass. Taken together, the results indicate that the vinyl ether linkage in the proximal portion of the sn-1 aliphatic chain of plasmenylcholine has substantial effects on the molecular dynamics of membrane bilayers both locally and at sites spatially distant from the covalent alteration.     

Microwave-assisted synthesis of deuterium labeled estrogen fatty acid esters

Deuterated analogs of estrogen fatty acid esters are needed as internal standards for isotope dilution GC/MS analyses. We have developed a rapid and efficient synthesis for 2,4,16,16-D4-estrone palmitate, stearate, oleate, linoleate, and linolenate and the corresponding 2,4,16,16,17alpha-D5-estradiol fatty acid 17-mono and 3,17-diesters using analogous fatty acid chlorides or fatty acid anhydrides and 4-(dimethylamino)pyridine under microwave irradiation. Chemoselective hydrolysis of fatty acid diesters was carried out by KOH in t-BuOH.     

New chemical trends in ganglioside research

A report is given of recent progress in the methodology for isolation of gangliosides from natural sources, for the preparation of molecular species of gangliosides homogeneous in both the oligosaccharide and ceramide portions of the molecule, for chemical manipulation and derivatization of gangliosides, and for the preparation of gangliosides radiolabelled in different parts of the molecule. Particular emphasis has been given to: high performance liquid chromatographic procedures capable to separate gangliosides on the basis of their oligosaccharide or ceramide moieties and yielding completely homogeneous compounds, that is gangliosides with a single oligosaccharide, a single long chain base and a single fatty acid; two-dimensional thin-layer chromatographic procedures, provided with a fully computerized quantification system, particularly suitable to identifying gangliosides containing alkali-labile linkages, including ganglioside lactones; chemical procedures of high yield for reducing gangliosides at the double bond of long chain base, for selective removal of the fatty acyl moiety and replacement with a novel fatty acid, and for the synthesis of ganglioside lactones; chemical procedures for inserting fluorescent, paramagnetic or photoreactive probes at the fatty acyl part of the ganglioside molecule; procedures for chemical isotopic radiolabelling of gangliosides at the level of sialic acid acetyl group and at the fatty acid moiety. Examples are provided evidencing the significance and potential use of a variety of ganglioside derivatives in the study of ganglioside metabolism and functional implications.     

A simple method for the synthesis of cholesterol esters in high yield.

A simple and convenient synthesis of cholesterol esters from cholesterol and fatty acid anhydrides is described. The high, reproducible yield of cholesterol ester and the small excess of fatty acid used makes this method attractive for the preparation of cholesterol esters using difficultly obtained fatty acids.     

Purification and characterization of canine myocardial cytosolic phospholipase A2. A calcium-independent phospholipase with absolute f1-2 regiospecificity for diradyl glycerophospholipids

Recently, we identified a novel calcium-independent, plasmalogen-selective phospholipase A2 activity in canine myocardial cytosol which represents the major measurable phospholipase A2 activity in myocardial homogenates (Wolf, R. A., and Gross, R. W. (1985) J. Biol. Chem. 260, 7295-7303). We now report the 154,000-fold purification of this phospholipase A2 to homogeneity through utilization of sequential anion exchange, chromatofocusing, affinity, Mono Q, and hydroxylapatite chromatographies. The purified enzyme had a molecular mass of 40 kDa, possessed a specific activity of 227 mumol/mg min, had a pH optimum of 6.4, and catalyzed the regiospecific cleavage of the sn-2 fatty acid from diradyl glycerophospholipids. The purified polypeptide was remarkable for its ability to selectively hydrolyze plasmenylcholine in homogeneous vesicles (subclass rank order: plasmenylcholine greater than alkyl-ether choline glycerophospholipid greater than phosphatidylcholine) as well as in mixed bilayers comprised of equimolar plasmenylcholine/phosphatidylcholine. Purified myocardial phospholipase A2 also possessed selectivity for hydrolysis of phospholipids containing arachidonic acid at the sn-2 position in comparison to oleic or palmitic acid. Taken together, these results constitute the first purification of a calcium-independent phospholipase with absolute regiospecificity for cleavage of the sn-2 acyl linkage in diradyl glycerophospholipids and demonstrate that myocardial phospholipase A2 has kinetic characteristics which are anticipated to result in the selective hydrolysis of sarcolemmal phospholipids during myocardial ischemia.     

Studies on the synthesis of rat liver fatty acid synthetase.

The synthesis of the multienzyme complex rat liver fatty acid synthetase was investigated utilizing modifications of methods developed in the laboratory of Schimke (Schimke, R. T. (1964) J. Biol. Chem. 239, 3808-3817 and Arias, I. M., Doyle, D., and Schimke, R. T. (1969) J. Biol. Chem. 244, 3303-3315). The relative amounts of radioactivity from a pulse of labeled lysine appearing in polypeptides derived from purified synthetase complex can be measured compensating for the varying amounts of lysine per polypeptide chain. The results show that labeled amino acid is incorporated into polypeptides derived from the complex at heterogeneous rates. However, 10 to 15 hours after the administration of a pulse, the amount of label per lysine residue in these polypeptides is identical. The results support the previously proposed model of this multienzyme complex (Tweto, J., Dehlinger, P., and Larrabee, A. R. (1972) Biochem. Biophys. Res. Commun. 48, 1371-1377). The previous work and that reported here suggests the existence of a pool of synthetase subunits which is an obligatory intermediate in both synthesis and turnover of the complex. The results obtained in this work are consistent with this model if the exchange of subunits into the intact complex is a relatively slow process requiring several hours to reach equilibrium.     

Human neutrophils incorporate arachidonic acid and saturated fatty acids into separate molecular species of phospholipids

The incorporation of radiolabeled arachidonic acid and saturated fatty acids into choline-linked phosphoglycerides (PC) of rabbit and human neutrophils was investigated by resolving the individual molecular species by reversed-phase high performance liquid chromatography. PC from neutrophils incubated with a mixture of [3H]arachidonic acid and [14C]stearic or [14C]palmitic acid contains both radiolabels; however, double labeling of individual molecular species is minimal. After labeling for 2 h, the [3H]arachidonate is distributed almost equally between diacyl and 1-O-alkyl-2-acyl species, but it is incorporated into diacyl species containing unlabeled stearate or palmitate at the sn-1 position. In contrast, labeled saturated fatty acids are incorporated only into diacyl species and contain predominantly oleate and linoleate at the sn-2 position. Labeled linoleate is not incorporated into ether-linked species, but is found in the same species as labeled stearate. The findings suggest that mechanisms exist in neutrophils for specific shunting of exogenous arachidonic acid into certain phospholipid molecular species and support the concept that the 1-O-alkyl-2-arachidonoyl species may be a functionally segregated pool of arachidonic acid within the PC of neutrophils.     

Synthesis of sugar fatty acid esters by modified lipase.

A simple synthesis of sugar fatty acid esters was developed in a nonaqueous solution using lipase modified by synthetic detergent. Esterification of sugar was accelerated by continuous removal of water from the reaction mixture with a molecular sieve. When glucose and palmitic acid (1:1 by mole) were used as the starting substrates, more than 90% of glucose was converted to its ester in this system. The resultant product was 6-O-palmitoylglucose. Other mono- or disaccharides were also esterified by the modified lipase with high yield. It was shown that the modified lipase might act as a catalyst for the synthesis of sugar fatty acid esters.     

Choline glycerophospholipid biosynthesis in the guinea pig heart

The aims of this study were to (i) elucidate the biosynthetic pathways for the formation of plasmenylcholine in the mammalian heart and (ii) investigate whether the control of choline glycerophospholipid production is different in hearts with high plasmenylcholine content. Guinea pig hearts were used throughout this study, since 34% of the cardiac choline glycerophospholipids in this species is present in the plasmenylcholine form. By perfusion of the guinea pig heart in the Langendorff mode with labeled choline, we demonstrated that the majority of plasmenylcholine in the heart was synthesized via the CDP-choline pathway. The ability of the heart to form plasmenylcholine from CDP-choline and 1-alkenyl-2-acylglycerol was also shown. We postulate that 1-alkenyl-2-acylglycerol in the guinea pig heart might originate from the hydrolysis of plasmenylethanolamine. In mammalian liver and other tissues, the CDP-choline pathway is the major pathway for phosphatidylcholine biosynthesis and the rate-limiting step is catalyzed by CTP:phosphocholine cytidylyltransferase. The results obtained from the present study support this supposition. In addition, evidence was obtained indicating that phosphorylation of choline by choline kinase in the CDP-choline pathway may also be rate limiting. Although the involvement of choline kinase as a rate-limiting enzyme in the CDP-choline pathway has been shown in a number of cell cultures, the rate-limiting role of this enzyme in intact mammalian organs has not been previously reported. The rationale for the presence of more than one rate-limiting step in the CDP-choline pathway in the guinea pig heart remains undefined.     

Chemical synthesis and NMR characterization of structured polyunsaturated triacylglycerols

The chemical synthesis of pure triacylglycerol (TAG) regioisomers, that contain long chain polyunsaturated fatty acids, such as arachidonic acid (AA) or docosahexaenoic acid (DHA), and saturated fatty acids, such as lauric acid (La) or palmitic acid (P), at defined positions, is described. A single step methodology using (benzotriazol-1-yloxy)-tripyrrolidinophosphonium hexafluorophosphate (PyBOP), an activator of carboxyl group commonly used in peptide synthesis and occasionally used in carboxylic acid esterification, has been developed for structured TAG synthesis. Identification of the fatty acyl chains for each TAG species was confirmed by atmospheric pressure chemical ionisation mass spectrometry (APCI-MS) and fatty acid positional distribution was determined by (1)H and (13)C NMR spectra. The generic described procedures can be applied to a large variety of substrates and was used for the production of specific triacylglycerols of defined molecular structures, with high regioisomeric purity. Combination of MS and NMR was shown to be an efficient tool for structural analysis of TAG. In particular, some NMR signals were demonstrated to be regioisomer specific, allowing rapid positional analysis of LC-PUFA containing TAG.     

All three promoters of the acetyl-coenzyme A-carboxylase alpha-encoding gene are expressed in mammary epithelial cells of ruminants.

The activity of the enzyme acetyl-CoA-carboxylase alpha (ACC-alpha) is rate limiting for the de novo synthesis of fatty acids. The encoding gene is expressed from three promoters in ruminants (PI-PIII). Their individual contribution to the formation of milk fat is unknown. Promoter-specific molecular probes were hybridized in situ to serial sections of mammary glands from cows and sheep to determine their developmental and spatial expression profile in the udder. We show that all three promoters are active in mammary epithelial cells (MECs) of udders from both species. This implies that, in principle, none of these promoters can be singled out as the key element controlling the ACC-alpha-related contribution to establishment of milk fat content, although the activity of PIII only is known to be disproportionally stimulated by lactation in MECs. We propose that all three promoters may be relevant for milk fat synthesis in cattle, whereas PII and PIII are crucial for milk fat formation in sheep. We show also that ACC-alpha synthesis is not strictly coupled to casein synthesis, particularly during pregnancy and involution.     

Nonradioactive labeling of probe with digoxigenin by polymerase chain reaction

Probes nonradioactively labeled with the steroid hapten digoxigenin have several intriguing properties, including a high sensitivity equivalent to that of radioactive probes, speed in detection, low hazard potential in handling, and possibility of long-term storage. The use of polymerase chain reaction for labeling probe has been demonstrated to offer various advantages including efficient labeling of fragments as small as 100 bp, direct labeling of genomic DNA, and labeling with subnanogram amounts of input DNA. We therefore investigated whether this technique could be adapted for labeling with a relatively large molecule such as digoxigenin. In this report, we show that the polymerase chain reaction is a very efficient technique for synthesis of digoxigenin-labeled DNA and we present an extremely simple procedure for purification of the non-isotopically labeled fragments.     

Microscale synthesis of dextran-based multivalent N-linked oligosaccharide probes

We developed a convenient method for the synthesis of dextran-based multivalent probes containing N-linked oligosaccharides which is efficient even in a small scale. Oligosaccharides were derivatized with succinic dihydrazide and dimethylamine borane under a mild acidic condition. The derivatized oligosaccharides were then conjugated in a good yield to periodate-oxidized dextran (500 kDa). Thus, the conjugates containing 120 to 140 oligosaccharide chains per dextran molecule were successfully synthesized. Their practical advantage was shown by the example that the asialofetuin oligosaccharide-dextran conjugate has much higher affinity to Ricinus communis agglutinin (RCA-I) than asialofetuin oligosaccharide itself or asialofetuin. The conjugates were further labeled with fluorescent reagent or biotinylation reagent containing a hydrazino group by the use of the unreacted aldehyde groups of the oxidized dextran, yielding probes with similar densities of fluorophores or biotin groups. Direct binding of the biotinylated asialofetuin oligosaccharide-dextran probe to RCA-I coated on the titer plate at a concentration of 50 ng/50 microl was easily detected using 50 fmol (as oligosaccharides) of the probe. The method for the synthesis of dextran-based oligosaccharide probes will facilitate the investigation of carbohydrate-mediated molecular interactions based on the native oligosaccharide structures.     

New approaches to synthesis of stereospecific sphingomyelin

Enormous progress in the asymmetric synthesis of stereochemically and chemically pure D-erythro-sphingosine and ceramides led to the development of a practical, efficient, easily scaleable process to provide industrial quantities of chiral sphingosine and ceramides. This established a new platform of chiral starting materials which facilitate the synthesis of complex sphingolipids. Utilizing stereochemically homogeneous, fully synthetic ceramides, two efficient synthetic methods were developed for the preparation of ultra pure stereochemically homogeneous sphingomyelins. The first method adapted highly efficient phosphoramidite technology from oligonucleotide chemistry. This method allows selective insertion of a phosphocholine moiety into 3-O-protected ceramide through phosphitylation, followed by choline attachment, phosphite oxidation and deprotection. This route provides stereochemically homogeneous sphingomyelin in 35-79% yield. The second route is based on the reaction of selectively protected ceramides with cyclic chlorophosphate followed by treatment with trimethylamine to give the desired sphingomyelins in 50% yield. Multigram quantities of 14C-labeled N-palmitoyl-D-erythro-sphingomyelin were produced with specific activity > 1000 dpm/nmol.