Phosphatidylcholine metabolism in endothelial cells: evidence for phospholipase A and a novel Ca2+-independent phospholipase C

The metabolism of phosphatidylcholine (PC) was investigated in sonicated suspensions of bovine pulmonary artery endothelial cells and in subcellular fractions using two PC substrates: 1-oleoyl-2-[3H]oleoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phospho[14C]choline. When these substrates were incubated with the whole cell sonicate at pH 7.5, all of the metabolized 3H label was recovered in [3H]oleic acid (95%) and [3H]diacylglycerol (5%). All of the 14C label was identified in [14C]lysoPC (92%) and [14C]phosphocholine (8%). These data indicated that PC was metabolized via phospholipase(s) A and phospholipase C. Substantial diacylglycerol lipase activity was identified in the cell sonicate. Production of similar proportions of diacylglycerol and phosphocholine and the low relative activity of phospholipase C compared to phospholipase A indicated that the phospholipase C-diacylglycerol lipase pathway contributed little to fatty acid release from the sn-2 position of PC. Neither phospholipase A nor phospholipase C required Ca2+. The pH profiles and subcellular fractionation experiments indicated the presence of multiple forms of phospholipase A, but phospholipase C activity displayed a single pH optimum at 7.5 and was located exclusively in the particulate fraction. The two enzyme activities demonstrated differential sensitivities to inhibition by p-bromophenacylbromide, phenylmethanesulfonyl fluoride and quinacrine. Each of these agents inhibited phospholipase A, whereas phospholipase C was inhibited only by p-bromophenacylbromide. The unique characteristics observed for phospholipase C activity towards PC indicated the existence of a novel enzyme that may play an important role in lipid metabolism in endothelial cells.     

Mixed-chain phosphatidylcholine bilayers: structure and properties

Calorimetric and X-ray diffraction data are reported for two series of saturated mixed-chain phosphatidylcholines (PCs), 18:0/n:0-PC and n:0/18:0-PC, where the sn-1 and sn-2 fatty acyl chains on the glycerol backbone are systematically varied by two methylene groups from 18:0 to 10:0 (n = 18, 16, 14, 12, or 10). Fully hydrated PCs were annealed at -4 degrees C and their multilamellar dispersions characterized by differential scanning calorimetry and X-ray diffraction. All mixed-chain PCs form low-temperature "crystalline" bilayer phases following low-temperature incubation, except 18:0/10:0-PC. The subtransition temperature (Ts) shifts toward the main (chain melting) transition temperature (Tm) as the sn-1 or sn-2 fatty acyl chain is reduced in length; for the shorter chain PCs (18:0/12:0-PC, 12:0/18:0-PC, and 10:0/18:0-PC), Ts is 1-2 degrees C greater than Tm, and the subtransition enthalpy (delta Hs) is much greater than for the longer acyl chain PCs. Tm decreases with acyl chain length for both series of PCs except 18:0/10:0-PC, while for the positional isomers, n:0/18:0-PC and 18:0/n:0-PC, Tm is higher for the isomer with the longer acyl chain in the sn-2 position of the glycerol backbone. The conversion from the crystalline bilayer Lc phase to the liquid-crystalline L alpha phase with melted hydrocarbon chains occurs through a series of phase changes which are chain length dependent. For example, 18:0/18:0-PC undergoes the phase changes Lc----L beta'----P beta'----L alpha, while the shorter chain PC, 10:0/18:0-PC, is directly transformed from the Lc phase to the L alpha phase. However, normalized enthalpy and entropy data suggest that the overall thermodynamic change, Lc----L alpha, is essentially chain length independent. On cooling, the conversion to the Lc phases occurs via bilayer gel phases, L beta', for the longer chain PCs or through triple-chain interdigitated bilayer gel phases, L beta, for the shorter chain PC 18:0/12:0-PC and possibly 10:0/18:0-PC. Molecular models indicate that the bilayer gel phases for the more asymmetric PC series, 18:0/n:0-PC, must undergo progressive interdigitation with chain length reduction to maintain maximum chain-chain interaction. The L beta phase of 18:0/10:0-PC is the most stable structure for this PC below Tm. The formation and stability of the triple-chain structures can be rationalized from molecular models.     

Loss of stereospecificity of phospholipases C and D upon introduction of a 2-alkyl group into rac-1,2-diacylglycero-3-phosphocholine

rac-1-[1-14C]Lauroyl-2-oleylglycero-3-phospho[methyl-3H]choline and rac-1-lauroyl-2-[1-14C]oleoylglycero-3-phospho[methyl-3H]choline along with rac-1-palmitoyl-2-oleylglycero-3-phosphocholine and sn-1-palmitoyl-2-oleylglycero-3-phosphocholine were synthesized and subjected to hydrolysis with phospholipase C (EC 3.1.4.3) from Clostridium perfringens and phospholipase D (EC 3.1.4.4) from cabbage. Kinetics of hydrolysis of the radioactive substrates were determined by measuring the 3H radioactivity retained in the aqueous phase due to free choline and phosphocholine and the 3H and 14C radioactivity recovered in the organic phase due to the released diacylglycerols and phosphatidic acids and the residual phosphatidylcholines. The rate of hydrolysis of the unlabelled substrates by phospholipase C was determined by thin-layer chromatography and gas-liquid chromatography of the methanolysis products. The relative initial rates of hydrolysis of sn-1,2,- and sn-2,3-enantiomers were 100-200:1 for phospholipase C and 40-50:1 for phospholipase D using rac-1-lauroyl-2-oleoylglycero-3-phosphocholine as the substrate. The substitution of the 2-acyl group by an alkyl group resulted in a loss of stereospecificity, which was partial for phospholipase C (relative rates equal to 8-13:1) and total for phospholipase D. There was a parallel dramatic decrease (500-1000-fold) in the initial rate of hydrolysis with phospholipase C but the activity of phospholipase D was only moderately reduced (18-fold). These findings are consistent with the earlier observed loss of the stereospecificity of lipoprotein lipase following introduction of a 2-alkyl group into triacylycerols, and point to a general unsuitability of 2-alkyl-linked acylglycerols as substrates for the assay of the stereospecificity of lipases, as well as for the isolation of enantiomeric 2-alkylacylglycerols by means of stereospecific lipases.     

Formation of diacylglycerol by a phospholipase D-phosphatidate phosphatase pathway specific for phosphatidylcholine in endothelial cells

The conversion of phosphatidylcholine (PC) to diacylglycerol (DAG) was studied in sonicated endothelial cells and in subcellular fractions in the presence of 0.05% Triton X-100 and 2 mM EDTA. DAG formation occurred predominantly in an organelle fraction that sedimented at 15,000 x g. In parallel reactions with exogenous 1-oleoyl-2-[3H]oleoyl-PC (sn-2-[3H]DOPC) and phosphatidyl[3H]choline ([choline-3H]PC), [3H]DAG was formed by a reaction pathway in which [3H]choline was the only product derived from [choline-3H]PC. [3H]Choline was not formed secondarily from [3H]glycerophosphocholine or [3H]phosphocholine. Small amounts of [3H]phosphatidate ([3H]PA) were isolated from reactions with sn-2-[3H]DOPC at short incubation times, and substantial PA phosphatase activity was demonstrated. These data, taken together, supported a phospholipase D-PA phosphatase pathway of DAG formation. Kinetic data established that the low ratio of [3H]PA/[3H]DAG formed in reactions with sn-2-[3H]DOPC was due to a 15-fold higher Vmax and 7-fold lower apparent Km of the PA phosphatase. The [3H]PA/[3H]DAG product ratio was increased by addition of unlabeled PA or by selective extraction of phospholipase D with Triton X-100. The characteristics of the phospholipase D indicated a unique enzyme. Activity was optimal in the presence of EDTA and was almost totally dependent upon Triton X-100. The pH profile displayed a peak at 7.0. Of particular significance was the stringent substrate specificity. Phosphatidylinositol was not hydrolyzed, and activities towards phosphatidylethanolamine and sphingomyelin were at most 30- to 50-fold lower than those towards PC. Phospholipase D and PA phosphatase were identified in a number of rat tissues and other cells. The highest activities of phospholipase D were present in lung and endothelial cells. Phospholipase D was partially purified from rat lung by Triton X-100 extraction and anion exchange chromatography. When linked with PA phosphatase, the phospholipase D could initiate a pathway of DAG formation that is highly specific for PC.     

Incorporation and remodeling of extracellular phosphatidylcholine with short acyl residues in Saccharomyces cerevisiae

The pem1/cho2 pem2/opi3 double mutant of Saccharomyces cerevisiae, which is auxotrophic for choline because of the deficiency in methylation activities of phosphatidylethanolamine, grew in the presence of 0.1 mM dioctanoyl-phosphatidylcholine (diC(8)PC). Analysis of the metabolism of methyl-(13)C-labeled diC(8)PC ((methyl-(13)C)(3)-diC(8)PC) by electrospray ionization tandem mass spectrometry (ESI-MS/MS) revealed that it was rapidly converted to (methyl-(13)C)(3)-PCs containing C16 or C18 acyl chains. (Methyl-(13)C)(3)-8:0-lyso-PC, (methyl-(13)C)(3)-8:0-16:0-PC and (methyl-(13)C)(3)-8:0-16:1-PC, which are the probable intermediate molecular species of acyl chain remodeling, appeared immediately after 5 min of pulse-labeling and decreased during the subsequent chase period. These results indicate that diC(8)PC was taken up by the pem1 pem2 double mutant and that the acyl chains of diC(8)PC were exchanged with longer yeast fatty acids. The temporary appearance of (methyl-(13)C)(3)-8:0-lyso-PC suggests that the remodeling reaction may consist of deacylation and reacylation by phospholipase activities and acyltransferase activities, respectively. The detailed analyses of the structures of (methyl-(13)C)(3)-8:0-16:0-PC and (methyl-(13)C)(3)-8:0-16:1-PC by MS/MS and MS(3) strongly suggest that most (methyl-(13)C)(3)-8:0-16:0-PCs have a C16:0 acyl chain at sn-1 position, whereas (methyl-(13)C)(3)-8:0-16:1-PCs have a C16:1 acyl chain at either sn-1 or sn-2 position in a similar frequency, implying that the initial C16:0 acyl chain substitution prefers the sn-1 position; however, the C16:1 acyl chain substitution starts at both sn-1 and sn-2 positions. The current study provides a pivotal insight into the acyl chain remodeling of phospholipids in yeast.     

Endothelial lipase releases saturated and unsaturated fatty acids of high density lipoprotein phosphatidylcholine

We assessed the ability of endothelial lipase (EL) to hydrolyze the sn-1 and sn-2 fatty acids (FAs) from HDL phosphatidylcholine. For this purpose, reconstituted discoidal HDLs (rHDLs) that contained free cholesterol, apolipoprotein A-I, and either 1-palmitoyl-2-oleoylphosphatidylcholine, 1-palmitoyl-2-linoleoylphosphatidylcholine, or 1-palmitoyl-2-arachidonylphosphatidylcholine were incubated with EL- and control (LacZ)-conditioned media. Gas chromatography analysis of the reaction mixtures revealed that both the sn-1 (16:0) and sn-2 (18:1, 18:2, and 20:4) FAs were liberated by EL. The higher rate of sn-1 FA cleavage compared with sn-2 FA release generated corresponding sn-2 acyl lyso-species as determined by MS analysis. EL failed to release sn-2 FA from rHDLs containing 1-O-1'-hexadecenyl-2-arachidonoylphosphatidylcholine, whose sn-1 position contained a nonhydrolyzable alkyl ether linkage. The lack of phospholipase A(2) activity of EL and its ability to liberate [(14)C]FA from [(14)C]lysophosphatidylcholine (lyso-PC) led us to conclude that EL-mediated deacylation of phosphatidylcholine (PC) is initiated at the sn-1 position, followed by the release of the remaining FA from the lyso-PC intermediate. Thin-layer chromatography analysis of cellular lipids obtained from EL-overexpressing cells revealed a pronounced accumulation of [(14)C]phospholipid and [(14)C]triglyceride upon incubation with 1-palmitoyl-2-[1-(14)C]linoleoyl-PC-labeled HDL(3), indicating the ability of EL to supply cells with unsaturated FAs.     

Sn-2-monoacylglycerol, not glycerol, is preferentially utilised for triacylglycerol and phosphatidylcholine biosynthesis in Atlantic salmon (Salmo salar L.) intestine

Pathways of lipid resynthesis in the intestine of fish are relatively unknown. Various reports have suggested the existence of both sn-1,3-specific (pancreatic) and non-specific (bile salt-activated) lipase activity operating on dietary triacylglycerol (TAG) in the intestinal lumen of fish during digestion. Thus, sn-2-monoacylglycerol (2-MAG) and glycerol, respective hydrolytic products of each lipase, are absorbed and utilised for glycerolipid synthesis in enterocytes via two alternative routes: monoacylglycerol (MAG) and glycerol-3-phosphate (G3P) pathways. Despite different precursors, both pathways converge at the production of sn-1,2-diacylglycerol (1,2-DAG) where TAG or phosphatidylcholine (PC) synthesis can occur. To elucidate the relative activities of MAG and G3P pathways in Atlantic salmon enterocytes, intestinal segments were mounted in Ussing chambers where equimolar mixtures of sn-2-oleoyl-[1,2,3-(3)H]glycerol (2-MAG) and [(14)C(U)]glycerol, plus unlabelled 16:0 and 18:2n-6 as exogenous fatty acid sources, were delivered in bile salt-containing Ringer solution to the mucosa. The MAG pathway predominated, over the G3P pathway, synthesizing ca. 95% of total TAG and ca. 80% of total PC after a 3 h incubation period at 10 degrees C. Further, the 1,2-DAG branch point into TAG or PC was polarised towards TAG synthesis (6:1) via the MAG pathway but more evenly distributed between TAG and PC (1:1) via the G3P pathway. Effect of long-chain saturated, monounsaturated and polyunsaturated fatty acids on the synthesized TAG/PC ratio was assessed by individually exchanging 16:0, 18:1n-9 or 18:2n-6, for 16:0+18:2n-6, in mucosal solutions. TAG synthesis was influenced considerably more than PC synthesis, via either pathway, by exogenous fatty acids utilised. 18:1n-9 significantly stimulated TAG synthesis via the MAG pathway yielding a TAG/PC ratio of 12:1. Alternatively, 18:2n-6 stimulated TAG synthesis the most via the G3P pathway (TAG/PC=4:1). 16:0 significantly attenuated TAG synthesis via either pathway. Micellar fatty acid species also significantly affected intestinal active transport mechanisms as shown by decreasing transepithelial potential (TEP) and short-circuit current (SSC) with increasing fatty acid unsaturation. The epithelial integrity was, however, not compromised after 3 h of exposure to any of the fatty acids. The implications of these findings on dietary fatty acid composition and enterocytic lipid droplet accumulation are discussed.     

A novel mechanism of diglyceride formation. 12-O-tetradecanoylphorbol-13-acetate stimulates the cyclic breakdown and resynthesis of phosphatidylcholine

12-O-Tetradecanoylphorbol-13-acetate (TPA) treatment of Madin-Darby canine kidney cells resulted in an increased incorporation of 32Pi and [methyl-3H]choline into choline-containing phosphoglycerides (PC). In pulse-chase experiments, TPA treatment caused an increased release of [methyl-3H]choline from the PC fraction of prelabeled cells. When cells were prelabeled with [3H]arachidonic acid and [14C]palmitic acid, TPA treatment resulted in an increased synthesis of 14C, 3H-diglycerides. Further studies were done to determine the relationship between PC breakdown and diglyceride synthesis. Cells were preincubated with ether-linked 1-O-[3H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine which was acylated to form 1-O-[3H]hexadecyl-2-acyl-sn-glycero-3-phosphocholine. Subsequent treatment of these cells with TPA resulted in an increased synthesis of 1-O-[3H]hexadecyl-2-acyl-sn-glycerol compared to cells not stimulated with TPA. These findings demonstrate that TPA stimulates PC turnover in Madin-Darby canine kidney cells and provide evidence for a novel mechanism of diglyceride formation.     

Phosphatidylcholine molecular species involved in Γ-linolenic acid biosynthesis in microsomes from borage seeds

Boraginaceae seeds are particularly rich in Γ -linolenic acid (6,9,12-octadecatrienoic acid, Γ -18:3). In microsomes, the analysis of phosphatidylcholine (PC) molecular species by HPLC led to identification of 15 different molecular species; among them 4 contained Γ -18:3, mostly at position 2 of sn -glycerol. Time courses of acylation and desaturation in PC molecular species were examined when [¹⁴C]oleoyl-CoA or [¹⁴C]linoleoyl-CoA was provided as substrates to isolated microsomes. With [¹⁴C]oleoyl-CoA or [¹⁴C]linoleoyl-CoA and in the absence of NADH, 3 main labelled PC molecular species were found: 18:2/[¹⁴C]18:1, 16:0/[¹⁴C]18:1 and 18:1/[¹⁴C]18:1. When NADH was present in the incubation medium, the fatty acids were progressively desaturated by the Δ12- and Δ6-desaturases successively (with [¹⁴C]oleoyl-CoA as precursor) or by the Δ6-desaturase alone (with [¹⁴C]linoleoyl-CoA as precursor). In both types of experiments, 7 final desaturation products in microsomes were evidenced; among them, 3 contained radioactive Γ -18:3, i.e . 18:2/[¹⁴C] Γ -18:3, 18:1/[¹⁴C] Γ -18:3 and 16:0/[¹⁴C] Γ -18:3. While the Δ12-desaturase had no specificity for position on the glycerol backbone, labelled Γ -linolenic acid was recovered exclusively in the sn -2 position.     

Subcellular site and mechanism of synthesis of disaturated phosphatidylcholine in alveolar type II cell adenomas.

The site of synthesis of 1,2-disaturated-(diacyl)-sn-glycero-3-phosphocholine (Sat2PC) in mouse alveolar type II cell adenomas has been studied by conducting pulse-chase experiments. Isolation of microsomal and lamellar body fractions from adenomas after a 20-min pulse with [methyl-3H]choline demonstrates that Sat2PC first appears in the microsomal fraction, and after a short lag subsequently appears in the lamellar body fraction. The kinetics of labeling of Sat2PC are consistent with the microsomal membranes functioning as the subcellular site of synthesis for this pulmonary surfactant phospholipid. Short term labeling experiments with [9,10-3H]palmitate demonstrate that this fatty acid is incorporated into the sn-2 position of Sat2PC at a faster rate than its incorporation into the sn-1 position. This finding indicates that the synthesis of Sat2PC occurs by a deacylation-reacylation mechanism.     

Evidence of luminal phosphatidylcholine secretion in rat ileum

BACKGROUND: Intestinal mucus not only facilitates substrate absorption, but also forms a hydrophobic, phosphatidylcholine (PC) enriched, barrier against luminal gut contents. METHODS: For evaluation of the origin of PC in intestinal mucus, we first analyzed the mucus PC in mice with absent biliary phospholipid secretion (mdr2 (-/-) mice) using electrospray ionization (ESI) tandem mass spectroscopy (MS/MS). Second, in situ perfused rat jejunum, ileum and colon were analyzed after i.v. bolus injections of 155 pmol [(3)H]-PC. Additional in vitro experiments were performed with isolated mucosal cells after incubation with the PC precursor [(3)H]-choline. RESULTS: In mdr2 (-/-) mice and control animals no significant quantitative difference in mucus PC was found, indicating that mucus PC is of intestinal and not biliary origin. In situ perfusion studies detected intestinal secretion of [(3)H]-PC, which was stimulated in presence of 2 mM taurocholate (TC). Secretion rates of [(3)H]-PC were highest in ileum (9.0+/-0.8 fmol h(-1)xcm(-1)), lower in jejunum (4.3+/-0.5) and minimal in colon (0.8+/-0.2). It compares to an intestinal secretion of native PC originating to 64% from bile, 9% from jejunum, 28% from ileum, and 1% from colon. Complementary in vitro studies showed 30-min secretion rates for [(3)H]-PC to be highest in enterocytes from ileum (26.5+/-5.3% of intracellular [(3)H]-PC) and jejunum (19.8+/-2.9%), and significantly lower in colonocytes (8.4+/-1.3%). CONCLUSION: PC in the intestinal mucus originates from secretion by ileal and jejunal enterocytes.     

Phosphatidylcholine transfer protein from bovine liver contains highly unsaturated phosphatidylcholine species

The phosphatidylcholine transfer protein (PC-TP) from bovine liver contains one molecule of non-covalently bound PC. In order to gain more insight into the physiological function of PC-TP, PC was extracted from bovine liver PC-TP and its molecular species composition identified by fast atom bombardment mass spectrometry. The prevailing molecular species were C18:0/C18:1-, C18:0/C18:2-, C18:OIC20:4-, C18:0120:5- and C18:OIC22:5-PC accounting for 85% of the PC species present. This molecular species composition is not representative for what is present in bovine liver where these species account for 43% of the total PC content [Montfoort et al. (1971) Biochim. Biophys. Acta 231, 335–342]. Another striking observation is that PC species carrying a palmitoyl chain at the sn-1 position are nearly absent, despite these species being abundantly present in bovine liver. This study suggests that PC-TP could play a role in the metabolism of highly unsaturated, stearoyl-containing PC species.     

Mimicry of phorbol ester responses by diacylglycerols. Differential effects on phosphatidylcholine biosynthesis, cell-cell communication and epidermal growth factor binding

The biosynthesis of phosphatidylcholine (PC) in HEL-37 cells was followed by measuring the incorporation of [32P]Pi into PC. Incorporation was stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA) and by the synthetic diacylglycerol, sn-1,2-dioctanoylglycerol (diC8), but not by sn-1-oleoyl-2-acetylglycerol or sn-1,2-dihexanoylglycerol (diC6). DiC8 was rapidly metabolised by HEL-37 cells to the corresponding PC and phosphatidic acid derivatives. diC8, diC6 and oleoylacetylglycerol effectively displaced [3H]phorbol-12,13-dibutyrate bound to a soluble cell extract from HEL-37 cells, but only diC8 was able to displace the labelled phorbol ester from prelabelled cells. TPA, diC8, diC6 and oleoylacetylglycerol were all effective inhibitors of 125I-labelled epidermal growth factor binding to, and gap junctional communication between, HEL-37 cells. It is concluded that only cell-permeable diacylglycerols stimulate PC biosynthesis which may therefore require interaction with membranes other than the plasma membrane.     

Synthesis of saturated phosphatidylcholine and phosphatidylglycerol by freshly isolated rat alveolar type II cells

Saturated phosphatidylcholine and phosphatidylglycerol are important components of pulmonary surface active material, but the relative contributions of different pathways for the synthesis of these two classes of phospholipids by alveolar type II cells are not established. We purified freshly isolated rat type II cells by centrifugal elutriation and incubated them with [1-14C]palmitate as the sole exogenous fatty acid in one series of experiments or with [9,10-3H]palmitate, mixed fatty acids (16:0, 18:1 and 18:2), and [U-14C]glucose in another series of experiments. Type II cells readily incorporated [1-14C]palmitate into saturated phosphatidic acid (55-59% of total phosphatidic acid), saturated diacylglycerol (82-87% of total diacylglycerol), saturated phosphatidylcholine (69-76% of total phosphatidylcholine), and saturated phosphatidylglycerol (55-59% of total phosphatidylglycerol). Saturated phosphatidic acid, diacylglycerol and phosphatidylglycerol were nearly equally labeled in the sn-1 and sn-2 positions, whereas saturated phosphatidylcholine was preferentially labeled in the sn-2 position. With [9,10-3H]palmitate and [U-14C]glucose, the labeling patterns of phosphatidic acid, diacylglycerol and phosphatidylglycerol were similar to each other but different from that of phosphatidylcholine. The glucose label was found predominantly in the unsaturated phosphatidylcholines at early times (3-10 min) and in the saturated phosphatidylcholines at later times (30-90 min). Similarly, the 3H/14C ratio was very high in saturated phosphatidylcholine and always above that in saturated diacylglycerol. We conclude that freshly isolated type II cells synthesize saturated phosphatidic acid, diacylglycerol, phosphatidylcholine and phosphatidylglycerol and that under our in vitro conditions the deacylation-reacylation pathway is important for the synthesis of saturated phosphatidylcholine but is less important for the synthesis of saturated phosphatidylglycerol. By the assumptions stated in the text during the pulse chase experiment de novo synthesis of saturated phosphatidylcholine from saturated diacylglycerol accounted for 25% of the total synthesis of saturated phosphatidylcholine.     

Phospholipase D in homogenates from HL-60 granulocytes: implications of calcium and G protein control

Occupancy of chemotactic peptide receptors leads to rapid initiation of phospholipase D (PLD) activity in intact dimethylsulfoxide-differentiated HL-60 granulocytes (Pai, J.-K, Siegel, M.I., Egan, R.W., and Billah, M.M. (1988) J. Biol. Chem. 263, 12472). To gain further insight into the activation mechanisms, PLD has been studied in cell lysates from HL-60 granulocytes, using 1-0-alkyl-2-oleoyl-[32P]phosphatidylcholine (alkyl-[32P]PC), 1-0-[3H]alkyl-2-oleoyl-phosphatidylcholine [( 3H]alkyl-PC) and [14C]arachidonyl-phospholipids as substrates. In the presence of Ca2+ and GTP gamma S, post-nuclear homogenates degrade alkyl-[32P]PC to produce 1-0-alkyl-[32P]phosphatidic acid (alkyl-[32P]-PA), and in the presence of ethanol, also 1-0-alkyl-[32P]phosphatidylethanol (alkyl-[32P]PEt). By comparing the 3H/32P ratios of PA and PEt to that of PC, it is concluded that PA and PEt are formed exclusively by a PLD that catalyzes both hydrolysis and transphosphatidylation between PC and ethanol. Furthermore, PC containing either ester- or ether-linkage at the sn-1 position is degraded in preference to phosphatidylethanolamine and phosphatidylinositol by PLD in HL-60 cell homogenates. It is concluded that HL-60 granulocytes contain a PC-specific PLD that requires both Ca2+ and GTP for activation.     

The binding of phosphatidylcholine to the phosphatidylcholine transfer protein: affinity and role in folding

Bovine liver phosphatidylcholine transfer protein (PC-TP) has been expressed in Escherichia coli and purified to homogeneity from the cytosol fraction at a yield of 0.45 mg PC-TP per 10 mg total cytosolic protein. In addition, active PC-TP was obtained from inclusion bodies. An essential factor in the activation of PC-TP was phosphatidylcholine (PC) present in the folding buffer. PC-TP from the cytosol contains phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) with a preference for the di-monounsaturated species over the saturated species as determined by fast atom bombardment mass spectrometry (FAB-MS). By incubation with microsomal membranes the endogenous PE and PG were replaced by PC. Relative to the microsomal PC species composition, PC-TP bound preferentially C16:0/C20:4-PC and C16:0/C18:2-PC (twofold enriched) whereas the major microsomal species C18:0/C18:1-PC and C18:0/C18:2-PC were distinctly less bound. PC-TP is structurally homologous to the lipid-binding domain of the steroidogenic acute regulatory protein (Nat. Struct. Biol. 7 (2000) 408). Replacement of Lys(55) present in one of the beta-strands forming the lipid-binding site, with an isoleucine residue yielded an inactive protein. This suggests that Lys(55) be involved in the binding of the PC molecule.     

Platelet-activating factor promotes arachidonate incorporation into phosphatidylinositol and phosphatidylcholine in neutrophils

Platelet-activating factor (1-0-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) promotes the incorporation of [1-14C]arachidonic acid most significantly into phosphatidylinositol (PI) and phosphatidylcholine (PC) during the early stages of guinea pig neutrophil-PAF interaction. The stimulation reached a maximum at 10(-7) M and started to decline at 10(-6) M. No changes in the mass of each phospholipid were detected in neutrophils challenged by PAF for 1 to 5 minutes. The stimulation by PAF on the formation of [14C]arachidonoyl-PC but not [14C]arachidonoyl-PI was dependent on the presence of external Ca2+. These results suggest that the increased acylation of PI and PC elicited by PAF is secondary to an increased deacylation of these phospholipids and the mechanisms by which PAF stimulates the deacylation of PI and PC may be different.     

Elucidation of the Biosynthesis of Eicosapentaenoic Acid in the Microalga Porphyridium cruentum (II. Studies with Radiolabeled Precursors)

In the course of the study of the biosynthesis of the fatty acid eicosapentaenoic acid (EPA) in the microalga Porphyridium cruentum, cells were pulse-labeled with various radiolabeled fatty acid precursors. Our data show that the major end products of the biosynthesis are EPA-containing galactolipids of a eukaryotic and prokaryotic nature. The prokaryotic molecular species contain EPA and arachidonic acid at the sn-1 position and C16 fatty acids, mainly 16:0, at the sn-2 positions, whereas in the eukaryotic species both positions are occupied by EPA or arachidonic acid. However, we suggest that both the eukaryotic and prokaryotic molecular species are formed in two pathways, [omega]6 and [omega]3, which involve cytoplasmic and chloroplastic lipids. In the [omega]6 pathway, cytoplasmic 18:2-phosphatidylcholine (PC) is converted to 20:4[omega]6-PC by a sequence that includes a [delta]6 desaturase, an elongation step, and a [delta]5 desaturase. In the minor [omega]3 pathway, 18:2-PC is presumably desaturated to 18:3[omega]3, which is sequentially converted by the enzymatic sequence of the [omega]6 pathway to 20:5[omega]3-PC. The products of both pathways are exported, as their diacylglycerol moieties, to the chloroplast to be galactosylated into their respective monogalactosyldiacylglycerol molecular species. The 20:4[omega]6 in both eukaryotic and prokaryotic monogalactosyldiacylglycerol can be further desaturated to EPA by a chloroplastic [delta]17 ([omega]3) desaturase.     

Structure and properties of mixed-chain phosphatidylcholine bilayers

The structural and thermotropic properties of the hydrated mixed-chain phosphatidylcholines (PCs), C(8):C(18)-PC and C(10):C(18)-PC, have been studied by X-ray diffraction and differential scanning calorimetry. For fully hydrated C(8):C(18)-PC, the reversible chain melting transition is observed at 9.9 degrees C (delta H = 7.3 kcal/mol). X-ray diffraction at 0 degrees C (below the chain melting transition) shows a small bilayer repeat distance, d = 51.0 A, and a sharp, symmetric wide-angle reflection at 4.1 A, characteristic of a mixed interdigitated bilayer gel phase [see McIntosh, T. J., Simon, S. A., Ellington, J. C., Jr., & Porter, N. A. (1984) Biochemistry 23, 4038-4044; Hui, S. W., Mason, J. T., & Huang, C. (1984) Biochemistry 23, 5570-5577]. At 30 degrees C (above the chain melting transition), a diffuse band is observed at 4.5 A characteristic of an L alpha phase but with an increased bilayer periodicity, d = 61 A. Both the calculated lipid bilayer thickness (d1) and that determined directly from electron density profiles (dp-p) show unusual increases as a consequence of chain melting. In contrast, fully hydrated C(10):C(18)-PC shows an asymmetric endothermic transition at 11.8 degrees C. Below the chain melting transition, two lamellar phases are present, corresponding to coexisting interdigitated (d = 52.3 A) and noninterdigitated (d = 62.5 A) bilayer gel phases. The relative amounts of these phases depend upon the low-temperature incubation and/or hydration conditions, suggesting conversions, albeit kinetically complex, between metastable, and stable phases. The different behavior of C(8):C(18)-PC and C(10):C(18)-PC, as well as their positional isomers, is rationalized in terms of the molecular conformation of PC.     

Molecular species of phosphatidylcholine in abetalipoproteinemia: effect of lecithin:cholesterol acyltransferase and lysolecithin acyltransferase

In order to study the role of very low density lipoproteins (VLDL) and low density lipoproteins (LDL) in determining the molecular species composition of phosphatidylcholine (PC) and the specificity of lecithin:cholesterol acyltransferase (LCAT) in human plasma, we studied the PC species composition in plasma from abetalipoproteinemic (ABL) and control subjects before and after incubation at 37 degrees C. The ABL plasma contained significantly higher percentages of sn-2-18:1 species (16:0-18:1, 18:0-18:1, and 18:1-18:1) and lower percentages of sn-2-18:2 species (16:0-18:2, 18:0-18:2, and 18:1-18:2) as well as sn-2-20:4 species (16:0-20:4, 18:0-20:4, and 18:1-20:4). Similar abnormalities were found in the PC of ABL erythrocytes, while the PE of the erythrocytes was less affected. The relative contribution of various PC species towards LCAT reaction in ABL plasma was significantly different from that found in normal plasma. Thus, while 16:0-18:2 and 16:0-18:1 contributed, respectively, 43.8% and 15.9% of the total acyl groups used for cholesterol esterification in normal plasma, they contributed, respectively, 21.5% and 37.9% in ABL plasma. The relative contribution of 16:0-20:4 was also significantly lower in ABL plasma (4.7% vs. 9.0% in normal), while that of 16:0-16:0 was higher (6.4% vs. 0.5%). However, the selectivity factors of various species (percent contribution/percent concentration) were not significantly different between ABL and normal plasma, indicating that the substrate specificity of LCAT is not altered in the absence of VLDL and LDL. Incubation of ABL plasma in the presence of normal VLDL or LDL resulted in normalization of its molecular species composition and in the stimulation of its LCAT activity. Addition of LDL, but not VLDL, also resulted in the activation of lysolecithin acyltransferase (LAT) activity. The incorporation of [1-14C]palmitoyl lysoPC into various PC species in the presence of LDL was similar to that observed in normal plasma, with the 16:0-16:0 species having the highest specific activity. These results indicate that the absence of apoB-containing lipoproteins significantly affects the molecular species composition of plasma PC as well as its metabolism by LCAT and LAT reactions.