Asymmetric <Emphasis Type="Italic">in vitro</Emphasis> synthesis of diastereomeric phosphatidylglycerols from phosphatidylcholine and glycerol by bacterial phospholipase D

Using chiral-phase HPLC, we determined the stereochemical configuration of the phosphatidylglycerols (PtdGro) synthesized in vitro from 1,2-diacyl-sn-glycero-3-phosphocholine (PtdCho, R configuration) or 1,2-diacyl-sn-glycero-3-phosphoethanolamine (PtdEtn, R configuration) and glycerol by transphosphatidylation with bacterial phospholipase D (PLD). The results obtained with PLD preparations from three Streptomyces strains (S. septatus TH-2, S. halstedii K5, and S. halstedii subsp. scabies K6) and one Actinomadura species were compared with those obtained using cabbage and peanut PLD. The reaction was carried out at 30°C in a biphasic system consisting of diethyl ether and acetate buffer. The resulting PtdGro were then converted into bis(3,5-dinitrophenylurethane) derivatives, which were separated on an (R)-1-(1-naphthyl)ethylamine polymer. In contrast to the cabbage and peanut PLD, which gave equimolar mixtures of the R,S and R,R diastereomers, as previously established, the bacterial PLD yielded diastereomixtures of 30–40% 1,2-diacyl-sn-glycero-3-phospho-1′-sn-glycerol (R,S configuration) and 60–70% 1,2-diacyl-sn-glycero-3-phospho-3′-sn-glycerol (R,R configuration). The highest disproportionation was found for the Streptomyces K6 species. The present study demonstrates that bacterial PLD-catalyzed transphosphatidylation proceeds to a considerable extent stereoselectively to produce PtdGro from PtdCho or PtdEtn and prochiral glycerol, indicating a preference for the sn-3′ position of the glycerol molecule.     

Effect of temperature on the stereoselectivity of phospholipase D toward glycerol in the transphosphatidylation of phosphatidylcholine to phosphatidylglycerol

In this study, the effect of temperature on the stereoselectivity of phospholipase D (PLD) toward the two primary hydroxyl groups of glycerol in the transphosphatidylation reaction of phosphatidylcholine to phosphatidylglycerol (PtdGro) was investigated. For this purpose, PLD from bacteria (Streptomyces septatus TH-2, S. halstedii subsp. scabies K6, and Actinomadura sp.) and cabbage were tested. At the reaction temperatures employed (0–60°C), the proportions of the two PtdGro diastereomers, namely, 1,2-dioleoyl-sn-glycero-3-phospho-3′-sn-glycerol (R,R configuration) and 1,2-dioleoyl-sn-glycero-3-phosphol-1′-sn-glycerol (R,S configuration), which were produced with PLD from Streptomyces TH-2 and Actinomadura sp., changed gradually from 50% R,R and 50% R,S at 50–60°C to 70% R,R and 30% R,S at O°C. These alterations suggested that the stereoselectivity of the bacterial PLD toward the two primary hydroxyl groups of prochiral glycerol was significantly influenced by reaction temperature. PLD from Streptomyces K6 showed relatively little effect of temperature on stereoselectivity, giving 65–69% R,R in the temperature range of 60–10°C examined. The plots of In ([R,R]/[R,S]) vs. 1/T gave good linear fits for these three bacterial PLD. No temperature effect was observed for cabbage PLD, which gave an almost equimolar mixture of the R,R and R,S diastereomers in the range from 0 to 40°C. The temperature-dependent change in enantiomeric selectivity of the bacterial PLD promises potentially profitable commercial exploitation.     

Synthesis of 6-phosphatidyl-L-ascorbic acid by phospholipase D

Phospholipase D (EC 3.1.4.4) ofStreptomyces species was found to catalyze transphosphatidylation to L-ascorbic acid from phosphatidylcholine (PC) in a biphasic reaction system. The product was identified as 1,2-diacyl-sn-glycero-3-phospho-6′-L-ascorbic acid (PA-AsA) by mass spectrometry and nuclear magnetic resonance spectroscopy. The optimal pH of transphosphatidylation was 4.5 and the rate of PA-AsA formation increased as concentrations of L-ascorbic acid increased. The conversion of PC to PA-AsA was greater than 80%. PA-AsA was found to be more resistant to hydrolysis by phospholipase D than was PC.     

Ether lipid content and fatty acid distribution in rabbit polymorphonuclear neutrophil phospholipids

This study was undertaken to determine if rabbit neutrophils contain sufficient ether-linked precursor for the synthesis of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activatin factor) by a deacylation-reacylation pathway. The phospholipids from rabbit peritoneal polymorphonuclear neutrophils were purified and quantitated, and the choline-containing and ethanolamine-containing phosphoglycerides were analyzed for ether lipid content. Choline-containing phosphoglycerides (37%), ethanolamine-containing phosphoglycerides (30%), and sphingomyelin (28%) were the predominant phospholipid classes, with smaller amounts of phosphatidylserine (5%) and phosphatidylinositol (<1%). The choline-linked fraction contained high amounts of 1-O-alkyl-2-acyl-(46%) and 1,2-diacyl-sn-glycero-3-phosphocholine (54%), with a trace of the 1-O-alk-1′-enyl-2-acyl species. The ethanolamine-linked fraction contained high amounts of 1-O-alk-1′-enyl-2-acyl-(63%) and 1,2-diacyl-sn-glycero-3-phosphoethanolamine (34%), and a low quantity of the 1-O-alkyl-2-acyl species (3%). The predominant 1-O-alkyl ether chains found in thesn-1 position of the choline-linked fraction were 16∶0 (35%), 18∶0 (14%), 18∶1 (26%), 20∶0 (16%), and 22∶0 (9%). The major 1-O-alk-1′-enyl ether chains found in thesn-1 position of the ethanolamine-linked fraction were 14∶0 (13%), 16∶0 (44%), 18∶0 (27%), 18∶1 (12%) and 18∶2 (3%). The major acyl groups in thesn-1 position of 1,2-diacyl-sn-glycero-3-phosphocholine and 1,2-diacyl-sn-glycero-3-phosphoethanolamine were 16∶0, 18∶0 and 18∶1. The most abundant acyl group in thesn-2 position of all classes of choline- and ethanolamine-linked phosphoglycerides was 18⩺2. Although this work does not define the biosynthetic pathway for platelet activating factor, it does show that there is ample precursor present to support its synthesis by a deacylation-reacylation pathway.     

Stereospecificity of monoacylglycerol and diacylglycerol acyltransferases from rat intestine as determined by chiral phase high-performance liquid chromatography

Using chiral phase high-performance liquid chromatography of diacylglycerols, we have redetermined the ratios of 1,2-/2,3-diacyl-sn-glycerols resulting from acylation of 2-monoacylglycerols by membrane bound and solubilized triacylglycerol systhetase of rat intestinal mucosa. With 2-oleoyl[-³H]glycerol as the acyl acceptor and oleoyl-CoA as the acyl donor, 97–98% of the diacylglycerol product was 1,2(2,3)-dioleoyl-sn-glycerol, 90% of which was thesn-1,2-and 10% thesn-2,3-enantiomer. The remaining diacylglycerol (less than 3%) was thesn-1,3-isomer. The overall yield of acylation products was 70%, of which 60% were diacylglycerols and 40% triacylglycerols. With 2-oleylglycerol ether as the acyl acceptor and [1-¹⁴C]oleoyl-CoA as the acyl donor, 90% of the diradylglycerol was 1-oleoyl-2-oleyl-sn-glycerol and 10% was the 2-oleyl-3-oleoyl-sn-glycerol. The diradylglycerols made up 96% and the triradylglycerols 4% of the radioactive product. With 1-palmitoyl-sn-glycerol as the acyl acceptor and [1-¹⁴C]oleoyl-CoA as the acyl donor, the predominant reaction product was 1-palmitoyl-3-oleoyl-sn-glycerol. The 3-palmitoyl-sn-glycerol was not a suitable acyl acceptor. Both 1,2- and 2,3-diacyl-sn-glycerols were substrates for diacylglycerol acyltransferase as neither isomer was favored when 1,2-dioleoyl-rac-[2-³H]glycerol was used as the acyl acceptor. There was a marked decrease in the acylation of the 1(3)-oleoyl-2-oleyl-sn-glycerol to the 1,3-dioleoyl-2-oleyl-sn-glycerol. It is concluded that neither monoacylglycerol nor diacylglycerol acyltransferase exhibit absolute stereospecificity for acylglycerols as fatty acid acceptors.     

Synthesis of a phosphatidyl derivative of vitamin E and its antioxidant activity in phospholipid bilayers

A novel phospholipid containing a chromanol structure at its polar head group was synthesized from egg yolk phosphatidylcholine and 2,5,7,8-tetramethyl-6-hydroxy-2-(hydroxyethyl)chroman by transphosphatidylation catalyzed by phospholipase D fromStreptomyces lydicus. The structure of the product synthesized was shown by spectral analysis to be 1,2-diacyl-sn-glycero-3-phospho-2′-hydroxyethyl-2′ 5′,7′,8′-tetramethyl-6′-hydroxychroman. The phosphatidylchromanol (PCh) showed antioxidant activity against radical chain oxidation of methyl linoleate in solution in a manner similar to that ofd-α-tocopherol (α-Toc) and 2,2,5,7,8-pentamethyl-6-chromanol. However, PCh was less effective as a chain-breaking antioxidant than was α-Toc when unilamellar egg yolk phosphatidylcholine liposomes were exposed to either a water-soluble or a lipid-soluble radical initiator. It is likely that the phospholipid nature of PCh affects the location and the mobility of the chromanol moiety in the membrane bilayer resulting in a decrease in antioxidant activity. On the other hand, the antioxidant activity of PCh was little different from that of α-Toc in unilamellar liposomes when exposed to a lipid-soluble radical initiator in the presence of ascorbic acid. It appears that PCh in phospholipid bilayers can be regenerated by ascorbic acid in aqueous phase as can be α-Toc. The new phospholipid, phosphatidylchromanol, should prove useful as a chain-breaking antioxidant in phospholipid membranes.     

Synthesis of C-18 mixed acid diacyl-sn-glycerol enantiomers

Procedures have been developed for the synthesis of both enantiomeric forms of mixed fatty acid, saturated and polyunsaturated 1,2-diacyl-sn-glycerols and 2,3-diacyl-sn-glycerols from D-mannitol as starting material. The following diacyl-sn-glycerols have been synthesized: 1-Stearoyl-2-linoleoyl-sn-glycerol, 1-stearoyl-2-linolenoyl-sn-glycerol, 2-linoleoyl-3-stearoyl-sn-glycerol and 2-linolenoyl-3-oleoyl-sn-glycerol. Their specific rotations, refractive indices, densities, solubilities, carbon and hydrogen analysis and iodine values have been reported. Presented in part at the ISF-AOCS World Congress, Chicago, September 1970.     

Inhibition of cardiolipin biosynthesis in the hypoxic rat heart

Cardiolipin is the principal polyglycerophospholipid in the heart. The effect of hypoxia on cardiolipin biosynthesis was investigated in isolated rat hearts perfused in the Langendorff mode. Hearts were pulsed-labeled for 60 min with 0.1 mM [1,(3)-³H]glycerol in Krebs Henseleit buffer saturated with either 95% O₂/5% CO₂ (control) or 95% N₂/5% CO₂ (hypoxic). Radioactivity incorporated into phosphatidylglycerol and cardiolipin were reduced 88% (P<.05) and 79% (P<.05), respectively, in hypoxic hearts compared to controls. In other experiments, hearts were pulse-labeled for 15 min with 1.4 mM [³²P]P_i in Krebs Henseleit buffer saturated with 95% O₂/5% CO₂ and subsequently perfused for 60 min under control or hypoxic conditions. The radioactivity incorporated into CDP-1,2-diacyl-sn-glycerol, phosphatidylglycerol, and cardiolipin were reduced 61% (P<.05), 71% (P<.05), and 70% (P<.05), respectively, in the hypoxic hearts compared to controls, indicating a decreased formation of CDP-1,2-diacyl-sn-glycerol in the hypoxic heart. The activities of the enzymes involved in cardiolipin biosynthesis and the cardiac pool sizes of cardiolipin, phosphatidylglycerol, and CDP-1,2-diacyl-1,2-diacyl-sn-glycerol were unaltered between hypoxic and control hearts. In contrast, cardiac adenosine-5′-triphosphate and CPT levels were decreased 94% (P<.05) and 92% (P<.05), respectively, in hypoxic hearts compared to controls. We postulate that the biosynthesis of the cardiac polyglycerophospholipid cardiolipin may be inhibited by a decreased adenosine-5′-triphosphate and cytidine-5′-triphosphate level in the heart.     

Conversion of Phosphatidylcholine to Phosphatidylglycerol with Phospholipase D and Glycerol

When phosphatidylcholine (PtdCho) is acted upon by the enzyme phospholipase D (PLD) in the presence of glycerol and water, at least two products can arise: phosphatidic acid (PtdOH) from hydrolysis and PtdGly from transphosphatidylation. Commercial PLD preparations from Streptomyces chromofuscus, Streptomyces sp., and cabbage were examined for their ability to selectively promote PtdGly formation in a two phase aqueous-organic solvent system. Factors examined were enzyme amount, pH, glycerol concentration, and the type of organic solvent. The reaction of PtdCho to give products such as PtdGly was followed by HPLC using a stationary phase consisting of a polymerized poly (vinyl alcohol) on silica gel with ELSD. The identities of all products were confirmed by retention times and HPLC-MS analyses. Under all tested conditions PLD from S. chromofuscus gave at most a 15% yield of PtdGly. Higher amounts of added glycerol inhibited this PLD. Nearly quantitative conversion to PtdGly was obtained with cabbage PLD when the mol ratio of glycerol to PtdCho was at least 64 (mol water/glycerol = 105). With PLD from Streptomyces sp. a nearly quantitative yield of PtdGly was obtained when the mol ratio of glycerol to PtdCho was at least 5.3 (mol water/glycerol = 1,266), demonstrating that this PLD had a higher selectivity for glycerol than cabbage PLD. When the glycerol concentration was very low, the level of PtdOH increased, and cardiolipin (diphosphatidylglycerol) was generated. The highest mol ratio PtdGly to PtdOH was observed when the solvents isopropyl ether or dichloromethane were used. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Role of lipid structure in the activation of phospholipase A2 by peroxidized phospholipids

The time course of hydrolysis of a mixed phospholipid substrate containing bovine liver 1,2-diacyl-sn-glycero-3-phosphocholine (PC) and 1,2-diacyl-sn-glycero-3-phosphoethanolamine (PE) catalyzed byCrotalus adamanteus phospholipase A₂ was measured before and after peroxidation of the lipid substrate. The rate of hydrolysis was increased after peroxidation by an iron/adenosine diphosphate (ADP) system; the presence of iron/ADP in the assay had a minimal inhibitory effect. The rate of lipid hydrolysis was also increased after the substrate was peroxidized by heat and O₂. Similarly, peroxidation increased the rate of hydrolysis of soy PC liposomes that did not contain PE. In order to minimize interfacial factors that may result in an increase in rate, the lipids were solubilized in Triton X-100. In mixtures of Triton with soy PC in the absence of PE, peroxidation dramatically increased the rate of lipid hydrolysis. In addition, the rate of hydrolysis of the unoxidizable lipid 1-palmitoyl-2-[1-¹⁴C]oleoyl PC incorporated into PC/PE liposomes was unaffected by peroxidation of the host lipid. These data are consistent with the notions that the increase in rate of hydrolysis of peroxidized PC substrates catalyzed by phospholipase A₂ is due largely to a preference for peroxidized phospholipid molecules as substrates and that peroxidation of host lipid does not significantly increase the rate of hydrolysis of nonoxidized lipids.     

Hydrolysis of neutral lipid substrates by rat hepatic lipase

Rat hepatic lipase, an enzyme whose involvement in the catabolism of lipoproteins remains poorly defined, has both neutral lipid and phospholipid hydrolyzing activity. We determined the substrate specificity of hepatic lipase for 1-oleoyl-sn-glycerol, 1,2-dioleoyl-sn-glycerol, and 1,3-dioleoyl-sn-glycerol in the Triton X-100 mixed micellar state, and compared these results to those obtained previously in our laboratory for the phospholipid substrates phosphatidic acid (PA), phosphatidylethanolamine (PE), and phosphatidylcholine (PC). V_max values were determined by diluting the substrate concentration in the surface of the micelle by Triton X-100. The V_max values obtained were 144 μmol/min/mg for 1-oleoyl-sn-glycerol, 163 μmol/min/mg for 1,2-dioleoyl-sn-glycerol, and 145 μmol/min/mg for 1,3-dioleoyl-sn-glycerol. These values were higher than those obtained earlier for phospholipids which were 67 μmol/min/mg for PA, 50 μmol/min/mg for PE and 4 μmol/min/mg for PC. In addition, the mole fraction of lipid substrate at half maximal velocity (K) in the surface dilution plot was lower for the neutral lipid substrates as compared to those obtained for the phospholipid substrates. When the hydrolysis of 1,3-dioleoyl-sn-glycerol mixed micelles was studied as a function of time, cleavage at thesn-1 andsn-3 positions occurred at the same rate, suggesting that hepatic lipase is not stereo-selective with respect to 1,3-diacyl-sn-glycerol substrates. To determine if the presence of one lipid could affect the hydrolysis of the other, all possible dual combinations of 1-oleoyl-sn-glycerol, 1,2-dioleoyl-sn-glycerol, and 1,3-dioleoyl-sn-glycerol, in the same micelle were made and the hydrolysis rate of each substrate was determined. Interaction occurred only for the 1,2-dioleoyl-sn-glycerol/1,3-dioleoyl-sn-glycerol mixture where the hydrolysis of 1,2-dioleoyl-sn-glycerol was slightly inhibited and that of 1,3-dioleoyl-sn-glycerol slightly activated compared to the predicted theoretical rate. These findings demonstrate that when presented in similar physical states, the neutral lipid substrates tested were hydrolyzed at a higher rate by hepatic lipase than the phospholipid substrates.     

1-O-alkyl-linked phosphoglycerides of human platelets: Distribution of arachidonate and other acyl residues in the ether-linked and diacyl species

In this study, the 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine content of human platelets was determined. The distribution of arachidonate among the 1,2-diacyl, 1-O-alkyl-2-acyl, and 1-O-alk-l′-enyl-2-acyl classes of choline- and ethanolamine-containing phosphoglycerides was also assessed. The major platelet phospholipids were choline-containing phosphoglycerides (38%), ethanolamine-containing phosphoglycerides (25%) and sphingomyelin (18%), with smaller amounts of phosphatidylserine (11%) and phosphatidylinositol (4%). In addition to the diacyl class, the choline-linked fraction was found to contain both 1-O-alkyl-2-acyl (10%) and 1-O-alk-l′-enyl-2-acyl (9%) species. The ethanolamine-linked fraction, on the other hand, had an elevated level of the 1-O-alk-l′-enyl-2-acyl (60%) species and a small amount of the 1-O-alkyl-2-acyl component (4%). The major fatty acyl residues found in all classes of the choline and ethanolamine phospholipids were 16∶0, 18∶0, (Δ⁹), 18∶2(n−6) and 20∶4(n−6). The 1-O-alk-l and 1-O-alk-l′-enyl fraction of the ethanolamine-linked phospholipids also contained substantial amounts of 22∶4(n−6), 22∶5(n−3) and 22∶6(n−3) acyl chains. Arachidonate comprised 44% of the acyl residues in thesn-2 position of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine. Corresponding values for the diacyl and 1-O-alk-l′-enyl-2-acyl species were 23% and 25%, respectively, based on all 20∶4(n−6) being linked to thesn-2 position of all classes. In the ethanolamine-linked phosphoglycerides, arachidonate constituted 60%, 20% and 68% of the acyl groups in thesn-2 position of the 1,2-diacyl, 1-O-alkyl-2-acyl and 1-O-alk-l′-enyl-2-acyl classes, respectively. The content of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine appears sufficient to support the synthesis of platelet activating factor by a deacylation-reacylation pathway in platelets. Our findings also demonstrate that human platelets contain a significant amount of 1-O-alkyl-2-arachidonyl-sn-glycero-3-phosphocholine that could possibly serve as a precursor of both platelet activating factor and bioactive arachidonate metabolites.     

Resolution of radiolabeled molecular species of phospholipid in human platelets: Effect of thrombin

Resolution of individual molecular species of human platelet 1,2-diradyl-sn-glycero-3-phosphocholines and 1,2-diradyl-sn-glycero-3-phosphoethanolamines by reverse phase high pressure liquid chromatography (HPLC) allowed a thorough analysis of those phospholipids labeled with [³H]arachidonic acid. Approximately 54% and 16% of the total incorporated radiolabel was found in choline glycerophospholipids and ethanolamine glycerophospholipids, respectively, with ca. 90% of this being found in the 1,2-diacyl molecular species. Eighty percent of [³H]-arachidonic acid incorporated into 1-acyl-2-arachidonoyl-sn-glycero-3-phosphocholine in resting platelets was equally distributed between 1-palmitoyl-2-arachidonoyl and 2-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, while 70% of the radiolabel in 1-acyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine was found in 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine. Thrombin stimulation (5 U/ml for 5 min) resulted in deacylation of all 1-acyl-2-[³H]arachidonoyl molecular species of 1-acyl-2-arachidonoyl-sn-glycero-3-phosphocholine and 1-acyl-2-arachidonoyl-sn-glycero-3-ethanolamine. There was also a slight increase in 1-O-alkyl-2-[³H]arachidonoyl-sn-glycero-3-phosphocholine and a significant increase in 1-O-alk-1′-enyl-2-[³H]arachidonoyl-sn-glycero-3-phosphoethanolamine molecular species of over 300%. Thus, HPLC methodology indicates that arachidonoyl-containing molecular species of phosphatidylcholine and phosphatidylethanolamine are the major source of arachidonic acid in thrombin-stimulated human platelets, while certain ether phospholipid molecular species become enriched in arachidonate.     

Reaction Specificity of Phospholipase D Prepared from Acinetobacter radioresistens a2 in Transphosphatidylation

Phospholipase D (PLD) can react with phospholipids as substrates, generally phosphatidylcholine (PtdCho), and the PLD‐substrate intermediate can be cleaved by another alcohol, resulting in transphosphatidylation of the substrate, which can be used in the production of special lipids. In this study, the reaction conditions affecting the transphosphatidylation of PtdCho with serine were optimized and the reaction specificity of a novel PLD prepared from Acinetobacter radioresistens a2 was evaluated for transphosphatidylation with a variety of phospholipid substrates and head group donors. Based on the yield of phosphatidylserine, experimental kinetic data, maximum transphosphatidylation rate, and kinetic constant, the specificity of PLD in transphosphatidylation was found to be affected by unsaturated fatty‐acid phospholipid substrates. The catalytic efficiency of PLD prepared from A. radioresistens a2 on the synthesis of natural phospholipids is on the order of l ‐serine > ethanolamine and glycerol ? inositol. Moreover, it was found that the transphosphatidylation of PtdCho with saccharides was related to the length of the carbon chain and the number of saccharide units.     

The Identification and Quantification of Phospholipids from Thermus and Meiothermus Bacteria

Structural identities of the major phospholipid (PL-2), minor phospholipid (PL-1) and trace phospholipid (PL-0) from representative strains of the genera Thermus and Meiothermus were established. Phospholipids were quantified using phosphorus-31 nuclear magnetic resonance (³¹P-NMR). The structures of the major phospholipid (PL-2) from Thermus filiformis MOK14.7 and Meiothermus ruber WRG6.9 were identified as 2′-O-(1,2-diacyl-sn-glycero-3-phospho)-3′-O-(α-N-acetylglucosaminyl)-N-glyceroyl alkylamine (GlcNAc-PGAA) and 2′-O-(2-acylalkyldiol-1-O-phospho)-3′-O-(α-N-acetylglucosaminyl)-N-glyceroyl alkylamine (GlcNAc-diolPGAA). Interestingly, M. ruber contained only a diacyl form of GlcNAc-PGAA (87 %), while T. filiformis contained both GlcNAc-PGAA (59 %) and GlcNAc-diolPGAA (18 %). The structures of the minor phospholipid (PL-1) were established as 2′-O-(1,2-diacyl-sn-glycero-3-phospho)-3′-O-(α-glucosaminyl)-N-glyceroyl alkylamine (GlcN-PGAA, 13 %) in T. filiformis and 2′-O-(1,2-diacyl-sn-glycero-3-phospho)-3′-O-(α-galactosaminyl)-N-glyceroyl alkylamine (GalN-PGAA, 19 %) in M. ruber. This is the first reliable discovery of phosphatidylglyceroylalkylamines modified by glucosamine or galactosamine with a free amino group. No signs of diol-based phosphatidylglyceroylalkylamines were found in PL-1 phospholipids. Similar to PL-2, trace phospholipid (PL-0) from T. filiformis contained both unsubstituted diol-based phosphatidylglyceroylalkylamine (diolPGAA) and PGAA, while M. ruber contained only free PGAA. Unlike analysis using TLC, the diol form of phosphatidylglyceroylalkylamines is clearly resolved from the diacyl form via ³¹P-NMR.     

Intestinal absorption of ester and ether glycerophospholipids in guinea pig. Role of a phospholipase A2 from brush border membrane

In vivo intestinal perfusion was used to follow the absorption of three different choline glycerophospholipids (CGP) in guinea pig. These included 1-[³H]palmitoyl-2-acyl-sn-glycero-3-phosphocholine (diacyl-GPC), 1-[³H]-O-hexadecyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl-GPC) and 1,2-di-O-hexadecyl-sn-glycero-3-phospho-[³H]-choline (dialkyl-GPC). About 80% of diacyl-GPC was absorbed within 4 hr, compared to 60% of alkylacyl-GPC and 30% of dialkyl-GPC. The radioactivity disappearing from the perfusion fluid was recovered in intestinal lipids, mostly triacylglycerol, free fatty acid and CGP from diacyl-GPC, CGP from alkylacyl-GPC and dialkyl-GPC. These results indicated that the nonhydrolyzable substrate dialkyl-GPC was much less absorbed, whereas diacyl-GPC, which released over 80% of [³H]palmitic acid in the perfusion fluid, displayed the highest absorption rate. The intermediate picture observed for alkylacyl-GPC suggested the possible involvement of a phospholipase A₂, which was detected in the entire intestinal tract. This enzyme was further found to concentrate in villus cells, where it is localized in the brush border membrane, as shown using two different subcellular fractionation procedures. These data suggest a possible role of this new enzyme in the digestion of alimentary phospholipids.     

Phosphatidylkojibiosyl diglyceride: Metabolism and function as an anchor in bacterial cell membranes

The recently discovered phosphoglycolipid, phosphatidylkojibiosyl diglyceride (PKD), was first observed as a biosynthetic by-product of glucosyl diglyceride metabolism inStreptococcus faecalis (faecium) ATCC 9790. Its structure is 1,2-diacyl-3-O(2′-O-α-D-glucopyranosyl-6′-O-phosphoryl-[1″,2″-diacyl-3″-O-sn-glycerol]-α-D-glucopyranosyl)-sn-glycerol. The biosynthesis of phosphatidylkojibiosyl diglyceride occurs by a novel transphosphatidylation reaction in which a phosphatidyl group is transferred from diphosphatidyl glycerol to the primary alcohol function at the 6 position of the internal glucose of kojibiosyl diglyceride. The reaction is catalyzed by a membrane-derived enzyme. Phosphatidylkojibiosyl diglyceride is bound covalently through a phosphodiester bond to the polyglycerol phosphate moiety of membrane lipoteichoic acid fromS. faecalis. Phosphatidylkojibosyl diglyceride has four nonpolar long chain fatty acyl groups and appears to have the necessary physico-chemical properties to anchor the long hydrophilic glycerol phosphate polymer of lipoteichoic acid to the hydrophobic environment of the membrane ofS. faecalis and probably other gram-positive bacteria as well.     

Critical analysis of phospholipid hydrolyzing activities in ripening tomato fruits. Study by spectrofluorimetry and high-performance liquid chromatography

Using the spectrofluorimetric method described by Wittenaueret al. [Wittenauer, L.A., Shirai, K., Jackson, R.L., and Johnson, J.D. (1984)Biochem. Biophys. Res. Commun. 118, 894–901] for phospholipase A₂ (PLA₂) measurement, we have detected a phospholipase activity in Ailsa Craig and in mutantrin tomatoes at their normal harvest time (mature green stage). This activity in Ailsa Craig tomatoes increased at the beginning of fruit ripening (green-orange stage) and then decreased slowly. The decrease in activity, however, was greater when ripening occurred after tomato picking at normal harvest time than when ripening occurred on tomato plants. This phospholipase activity was always higher inrin tomatoes than in normal ones. Thin-layer chromatography of compounds obtained after incubation of tomato extract demonstrated a decrease in the substrate 1-acyl-2-{6[(7-nitro-2,1,3, benzoxadiazol-4-yl)amino]-caproyl}-sn-glycero-3-phosphocholine (C₆-NBD-PC), and an increase in one product (NBD-aminohexanoic acid), but failed to detect the second product (1-acyl-sn-glycero-3-phosphocholine). We, therefore, developed a new one-step method for separation and quantification of a mixture of phospholipids and other lipids, using straight-phase-high-performance liquid chromatography with light-scattering detection. This method detected another fatty acid-releasing activity in enzyme extract from green-orange tomatoes. This lipolytic enzyme (or family of enzymes) slowly produced free fatty acids when 1-oleoyl-sn-glycero-3-phosphocholine was added as substrate. The production of fatty acids was stoichiometric and more rapid when 1-oleoyl-sn-glycero-3-phosphate and 1-oleoyl-sn-glycerol were used as substrates. On the other hand, the same tomato extract was unable to hydrolyze 1,2-dioleoyl-sn-glycero-3-phosphate and 1,2-dioleoyl-sn-glycerol. Crude tomato extract exhibited lipid acyl hydrolase activity according to the definition of Galliard [Galliard, T. (1979), inAdvances in the Biochemistry and Physiology of Plant Lipids (Appelqvist, L.A., and Liljenberg, C. eds.), pp. 121–132, Elsevier, Amsterdam]. But in order to demonstrate whether tomato extract contains PLA₂ activity and/or lysophospholipase activity, further work on purified tomato extract will be necessary.     

Synthesis of trideuteratedO-alkyl platelet activating factor and lyso derivatives

Racemic heavy isotope analogs of 1-O-alkyl-sn-glycero-3-phosphocholine (lysoPAF) and 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine (PAF) were prepared for use as internal standards to facilitate quantitative studies based on mass spectrometry. Starting from pentadencane-1,15-diol andrac-glycerol-1,2-acetonide, a convergent synthesis of 1-O-[16′-²H₃]hexadecyl and 1-O-[18′-²H₃]octadecylrac-glycero-3-phosphocholine and their acetyl derivatives is described. Three deuterium atoms were introduced at the terminal position of the 1-O-alkyl group by displacement of thep-toluensulfonyl group from 1-O-alkyl-15′-p-toluensulfonate and 1-O-alkyl-17′-p-toluensulfonate with [²H₃]-methylmagnesium iodide. The 1-O-alkyl-17′-p-toluensulfonate was obtained by reaction of the 1-O-alkyl-15′-p-toluensulfonate with allylmagnesium bromide, followed by reductive ozonolysis and treatment withp-toluenesulfonyl chloride. The hydroxyl group at C-2 was protected by a benzyl group and removed at a late stage in the synthesis. This provided the corresponding lysoderivatives or allowed preparation of racemic PAF by subsequent acetylation of the free hydroxy group. The phosphocholine moiety was introduced at glycerol C-3 by reaction with bromoethyldichlorophosphate and trimethylamine. The synthetic compounds were analyzed by FAB/MS and GC/NICIMS. They were shown to contain less than 0.6% protium impurity.     

Reversibility of cholinephosphotransferase in lung microsomes

The effect of cytidine 5′-monophosphate (CMP) on the incorporation of cytidine 5′-diphosphate (CDP) [methyl-¹⁴C]choline or [1-¹⁴C]dipalmitoylglycerol into phosphatidylcholine (PC) catalyzed by rabbit lung microsomal CDP choline:1,2-diacyl-sn-glycerol cholinephosphotransferase (EC 2.7.8.2) was studied. In the presence of 0.85 mM CMP and nonsaturating diacylglycerol concentration, the incorporation of CDP[¹⁴C]choline into PC was markedly stimulated, but the incorporation of [¹⁴C]dipalmitoylglycerol into PC was inhibited. This was due to the increase of endogenous diacylglycerol generated from microsomal PC by the cholinephosphotransferase reverse reaction. However, the newly synthesized PC was not readily hydrolyzed in the presence of CMP. The results of this study suggest that the endogenous membranous diacylglycerol is utilized more preferentially for PC synthesis than the exogenous diacylglycerol and that the newly synthesized PC could rapidly equilibrate with the endogenous membrane PC pool.