Biosynthesis of pulmonary surfactant: Comparison of 1-palmitoyl-sn-glycero-3-phosphocholine and palmitate as precursors of dipalmitoyl-sn-glycero-3-phosphocholine in adenoma alveolar type II cells

Urethan-induced pulmonary adenomas of mice are composed of cells that appear to be morphologically identical to alveolar type II cells and synthesize disaturated diacyl-sn-glycero-3-phosphocholine, the major component of pulmonary surfactant. 1-[1-¹⁴C]Palmitoyl-sn-glycero-3-phosphocholine and [1-¹⁴C]palmitic acid were compared as precursors of disaturated diacyl-sn-glycero-3-phosphocholine in the adenoma type II cells by incubating both substrates with whole adenomas. When the precursors were compared at equal concentrations (100 μm) in the presence of albumin (1 mg/ml), the rates of incorporation of 1-[1-¹⁴C]palmitoyl-sn-glycero-3-phosphocholine and [1-¹⁴C]palmitic acid into diacyl-sn-glycero-3-phosphocholine were 5.2 and 2.9 nmol/min · g tissue, respectively. The concentration of monoacyl-sn-glycero-3-phosphocholine (lysolecithin) in the blood plasma of BALB/c mice was 150 μm. In short-term labeling experiments, the label in disaturated diacyl-sn-glycero-3-phosphocholine was equally distributed between the sn-1 and sn-2 positions when 1-[1-¹⁴C]palmitoyl-sn-glycero-3-phosphocholine was the precursor, whereas 75 to 80% was in the sn-2 position when [1-¹⁴C]palmitic acid was the precursor. The ratios are consistent with incorporation of 1-palmitoyl-sn-glycero-3-phosphocholine via the lysolecithin:lysolecithin transacylase reaction and incorporation of palmitate via acylation of 1-palmitoyl-sn-glycero-3-phosphocholine by acyl-CoA:lysolecithin acyltransferase. 1-[1-¹⁴C]Palmitoyl-sn-glycero-3-phospho-[³H-methyl]choline was incorporated into total cellular diacyl-sn-glycero-3-phosphocholine with an isotope ratio similar to that of the precursor; the disaturated species was more enriched in ¹⁴C. These findings indicate the cells take up intact monoacyl-sn-glycero-3-phosphocholine and incorporate it into diacyl-sn-glycero-3-phosphocholine. The ability of the cells to utilize intact lysophosphoglycerides for synthesis of cellular lipids was further demonstrated by showing that ether analogs, 1-alkyl-sn-glycero-3-phosphocholine and 1-alkyl-sn-glycero-3-phosphoethanolamine, are taken up and acylated by the cells. Activities of lysolecithin:lysolecithin transacylase and acyl-CoA:lysolecithin acyltransferase were measured in subcellular fractions of the adenoma type II cells; the specific activities of the enzymes were 2.1 nmol/min · mg soluble protein and 21 nmol/min · mg microsomal protein, respectively. The total activity of the acyltransferase in the cell fractions was about four-fold higher than the activity of the transacylase. Characteristics of the two enzymes were studied and are discussed. The findings indicate that exogenous 1-palmitoyl-sn-glycero-3-phosphocholine and palmitic acid both serve as efficient precursors of disaturated diacyl-sn-glycero-3-phosphocholine in the adenoma alveolar type II cells.     

Lysophospholipase and lysophosphatidylcholine:Lysophosphatidylcholine transacylase from rat lung: Evidence for a single enzyme and some aspects of its specificity

An enzyme preparation was isolated from rat lung cytosol with the capability to transfer the fatty acyl chain from 1-acyl-sn-glycero-3-phosphocholine to water and to another molecule of 1-acyl-sn-glycero-3-phosphocholine. The evidence presented to indicate that a single protein confers both activities includes: (a) both normal and sodium dodecyl sulfate polyacrylamide disc gel electrophoresis showed a single protein band, and (b) heat treatment and preincubation with increasing amounts of diisopropylfluorophosphate resulted in concomitant loss of fatty acid and phosphatidylcholine formation. The enzyme converted 1-[9,10-³H₂]stearoyl-sn-glycero-3-phospho[¹⁴C-methyl]choline into phosphatidylcholine with an isotopic ³H/¹⁴C ratio twice that of the substrate, even when an excess of unlabeled fatty acid was present. The acyl group from palmitoyl-propanediol (1,3)-phosphocholine and palmitoyl-propanediol (1,3)-phosphoethanolamine could be transferred to lysophosphatidylcholine acceptor to yield phosphatidylcholine. Neither acylglycerols and cholesterol nor glycero-3-phosphate and glycero-3-phosphocholine served as acyl acceptors. Lysophosphatidylethanolamine and lysophosphatidyglycerol were converted also into the corresponding diacylphospholipids. Palmitoyllysophosphatidylcholine is preferentially converted into phosphatidylcholine when compared with stearoyllysophosphatidylcholine. The possible involvement of the enzyme in the synthesis of dipalmitoylphosphatidylcholine for the production of lung surfactant is discussed.     

Characterization of the transacylase activity of rat liver 60-kDa lysophospholipase-transacylase. Acyl transfer from the sn-2 to the sn-1 position

Rat liver 60-kDa lysophospholipase-transacylase catalyzes not only the hydrolysis of 1-acyl-sn-glycero-3-phosphocholine, but also the transfer of its acyl chain to a second molecule of 1-acyl-sn-glycero-3-phosphocholine to form phosphatidylcholine (H. Sugimoto, S. Yamashita, J. Biol. Chem. 269 (1994) 6252–6258). Here we report the detailed characterization of the transacylase activity of the enzyme. The enzyme mediated three types of acyl transfer between donor and acceptor lipids, transferring acyl residues from: (1) the sn-1 to -1(3); (2) sn-1 to -2; and (3) sn-2 to -1 positions. In the sn-1 to -1(3) transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1(3) positions of glycerol and 2-acyl-sn-glycerol, producing 1(3)-acyl-sn-glycerol and 1,2-diacyl-sn-glycerol, respectively. In the sn-1 to -2 transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to not only the sn-2 positions of 1-acyl-sn-glycero-3-phosphocholine, but also 1-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. 1-Acyl-sn-glycero-3-phospho-myo-inositol and 1-acyl-sn-glycero-3-phosphoserine were much less effectively transacylated by the enzyme. In the sn-2 to -1 transfer, the sn-2 acyl residue of 2-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1 position of 2-acyl-sn-glycero-3-phosphocholine and 2-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. Consistently, the enzyme hydrolyzed the sn-2 acyl residue from 2-acyl-sn-glycero-3-phosphocholine. By the sn-2 to -1 transfer activity, arachidonic acid was transferred from the sn-2 position of donor lipids to the sn-1 position of acceptor lipids, thus producing 1-arachidonoyl phosphatidylcholine. When 2-arachidonoyl-sn-glycero-3-phosphocholine was used as the sole substrate, diarachidonoyl phosphatidylcholine was synthesized at a rate of 0.23 μmol/min/mg protein. Thus, 60-kDa lysophospholipase-transacylase may play a role in the synthesis of 1-arachidonoyl phosphatidylcholine needed for important cell functions, such as anandamide synthesis.     

Comparison of 1-O-alkyl-, 1-O-alk-1'-enyl-, and 1-O-acyl-2-acetyl-sn-glycero-3-phosphoethanolamines and -3-phosphocholines as agonists of the platelet-activating factor family

Four naturally occurring platelet-activating factor (PAF) analogs, 1-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphocholine, 1-hexade-canoyl-2-acetyl-sn-glycero-3-phosphocholine, 1-octadecanoyl-2-acetyl-sn-glycero-3-phosphocholine, and 1-alkyl-2-acetyl-sn-glycero-3-phosphoethanolamine, stimulated human neutrophils (PMN) to mobilize Ca²⁺, degranulate, and produce Superoxide anion. They were, respectively, 5-, 300-, 500-, and 4000-fold weaker than PAF in each assay; inhibited PMN-binding of [³H]PAF at concentrations paralleling their biological potencies; and showed sensitivity to the inhibitory effects of PAF antagonists. PAF and the analogs, moreover, desensitized PMN responses to each other but not to leukotriene B₄ and actually increased (or primed) PMN responses to N-formyl-MET-LEU-PHE. Finally, 5-hydroxyicosatetraenoate-enhanced PMN responses to PAF and the analogs without enhancing the actions of other stimuli. It stereospecifically raised each analog's potency by as much as 100-fold and converted a fifth natural analog, 1-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine from inactive to a weak stimulator of PMN. PAF and its analogs thus represent a structurally diverse family of cell-derived phospholipids which can activate, prime, and desensitize neutrophils by using a common, apparently PAF receptor-dependent mechanism.     

On the phospholipid metabolism of glial cell primary cultures

Primary cultures prepared from newborn rat brain, consisted after 16 or 17 days mainly of astrocytes and of oligodendrocytes. 1-Alkenyl-sn-glycero-3-phosphoethanolamine (lysoplasmalogen) was used as substrate for studies on the metabolism of ethanolamine-glycerophospholipids. After 3 hr incubation two main products were observed: a) 1-alkenyl-2-acyl-sn-glycero-3-phosphoethanolamine (=ethanolamine plasmalogen) and b) 1-alkenyl-2-acyl-sn-glycero-3-phosphocholine (=choline plasmalogen). The acylation rate reached saturation at about 10 nmol substrate/mg cell protein with aV _max of 30 nmol×mg cell protein^–1×3 hr^–1. This acylated compound amounted to almost 60% of all radioactivity internalized, whereas the second product, choline plasmalogen, came to 20%. Unchanged substrate was found within the cells only in small amounts, even at maximum substrate internalization. These results were discussed in comparison with those obtained with 1-alkyl-sn-glycero-3-phosphoethanolamine under the same conditions (25).     

Mitochondrial H+-ATPase: Identification of Subunits of the F0 Subcomplex that Contact Membrane Lipids

The subunits of the F₀ membrane sector of bovine heart mitochondrial H⁺-ATPase that contact the lipids of the mitochondrial inner membrane were identified with the use of specially synthesized proteoliposomes that contained active mitochondrial H⁺-ATPase and a photoreactive lipid, which was 1-acyl-2-[12-[di-azocyclopentadiene-2-carbonylamino)-[12-¹⁴C]dodecanoyl]-sn-glycero-3-phosphocholine, 1-acyl-2-[11-([¹²⁵I]diazoiodocyclopentadiene-2-carbonyloxy)undecanoyl]-sn-glycero-3-phosphocholine, or 1-acyl-2-[12-(diazocyclopentadiene-2-carbonylamino)dodecanoyl]-sn-glycero-3-phosphocholine, where acyl is a mixture of the residues of palmitic (70%) and stearic (30%) acids. An analysis of the cross-linked products obtained upon the UV-irradiation of these proteoliposomes indicated that subunits c and a of the F₀ membrane sector contact the lipids. The crosslinked products were identified by SDS-PAGE and MALDI mass spectrometry.     

Characterization of the enzymatic hydrolysis of acetate from alkylacetylglycerols in the de novo pathway of PAF biosynthesis

This report describes the partial characterization of the enzymatic activity responsible for the hydrolysis of acetate from 1-alkyl-2-acetyl-sn-glycerol, the immediate precursor in the de novo synthesis of PAF (platelet-activating factor or 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by Ehrlich ascites cells. The highest acetylhydrolase activity for this neutral lipid was associated with the membrane fractions from Ehrlich ascites cells (> 90% of total activity); only a minimal level of activity (< 10%) was observed in the cytosol which contrasts with the cytosolic site of PAF acetylhydrolase in normal cells. Hydrolysis of 1-[³H]hexadecyl-2-acetyl-sn-glycerol by the membrane fraction at pH 7.5 and 37°C gave apparent values for K_m and V_max of 45 μM and 179 nmol/min per mg protein, respectively. Hydrolysis of acetate from 1-[³H]hexadecyl-2-acetyl-sn-glycerol by the membrane fraction was not affected by 5 mM concentrations of Ca⁺², Mg⁺² or EDTA, but was significantly inhibited (80% reduction) by 10 mM NaF. Based on differences in both the subcellular distribution and response to inhibition by NaF, the neutral lipid acetylhydrolase does not appear to be the same enzyme that hydrolyzes acetate from platelet-activating factor. In contrast to inhibition of diacylglycerol lipase by p-chloromercuribenzoate and N-ethylmaleimide, we found no significant inhibition of acetate hydrolysis from 1-[³H]hexadecyl-2-acetyl-sn-glycerol by either of these compounds. Also, p-nitrophenyl acetate (a nonspecific esterase substrate) failed to inhibit acetate hydrolysis of 1-[³H])hexadecyl-2-acetyl-sn-glycerol. Our studies of this enzyme would indicate that it may play an important role in regulating the levels of platelet-activating factor synthesized by the de novo pathway via hydrolysis of the immediate precursor of PAF.     

Bradykinin-stimulated release of [3H]arachidonic acid from phospholipids of HSDM1C1 cells: Comparison of diacyl phospholipids and plasmalogens as sources of prostaglandin precursors

Ethanolamine plasmalogens (1-alk-1′-enyl-2-acyl-sn-glycero-3-phosphoethanolamines) of many tissues contain high levels of arachidonate at their 2-position, and in certain tissues have been implicated as possible donors of arachidonate required in the synthesis of prostaglandins and thromboxanes. In the present study, [³H]arachidonate-labeled phospholipids of HSDM₁C₁ cells, a cell line derived from a mouse fibrosarcoma, were examined to determine the donor of the arachidonic acid released upon bradykinin stimulation of the synthesis of PGE₂. HSDM₁C₁ cells labeled with [³H]arachidonic acid for 24 hr in serum-free medium were used in most of the experiments and had the following distribution of label among the cellular lipids; phosphatidylcholine (33%), phosphatidylinositol (20%), diacyl-sn-glycero-3-phosphoethanolamine (15%), ethanolamine plasmalogen (15%), and less polar lipids (16%). Bradykinin treatment stimulated a rapid hydrolysis of [³H]arachidonate from the cellular lipids and conversion of the released acid to PGE₂, which was secreted into the medium. The label was released predominantly from phosphatidylinositol and possibly from phosphatidylcholine with no detectable change in the labeling of diacyl- or 1-alk-1′-enyl-2-acyl-sn-glycero-3-phosphoethanolamine. The ethanolamine plasmalogens, therefore, do not appear to be involved in the stimulated release of arachidonate in the HSDM₁C₁ cells. Indomethacin blocked the bradykinin-stimulated synthesis of PGE₂ and to a lesser degree inhibited the release of [³H]-arachidonate from the cellular lipids into the medium.     

In vivo metabolism of a new class of biologically active phospholipids: 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine,a platelet activating-hypotensive phospholipid

This report describes the in vivo metabolism of a new class of naturally occurring biologically active phospholipids (1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholines) that can cause hypotension and platelet aggregation. After intravenous injection in male rats, the acetylated ether phospholipid (1-[1′,2′-³H]alkyl) is rapidly cleared ($\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{?30}\text{s}$) from blood and its metabolites are found in a variety of tissues. The tissues containing the highest levels of radioactivity are lung, liver, spleen, and kidney. Chromatographic results showed that a considerable portion of the active lipid is not readily catabolized in some of the major tissues examined; however, inactive metabolites were also found, mainly 1-alkyl-2-lyso-$\text{sn}$-glycero-3-phosphocholine and 1-alkyl-2-acyl-$\text{sn}$-glycero-3-phosphocholine; the latter has a long chain fatty acid at the $\text{sn}$-2 position instead of the acetate. The findings are consistent with our earlier data that show these same tissues have the most active enzyme systems for metabolizing 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine.     

Continuous binding of the PAF molecule to its receptor is necessary for the long-term aggregation of platelets

Human and rabbit platelets fully aggregated byplatelet-activating factor (PAF) underwent slow disaggregation but wererapidly disaggregated by the PAF receptor antagonists WEB-2086,Y-24180, SM-12502, and CV-3988. Whereas the1-alkyl-2-[³H]acetyl-sn-glycero-3-phosphocholine([³H]acetyl-PAF)specifically bound to platelet receptors underwent slow and spontaneousdissociation, it dissociated promptly from its receptor when WEB-2086was added, in parallel with platelet disaggregation and disappearanceof P-selectin on the cell surface. Extracellular[³H]acetyl-PAF wasrapidly deacetylated by normal rabbit platelets; some of the[³H]acetyl-PAF wasbound to the cells and a very small amount of [³H]acetate wasdetected in the cells. In contrast, when1-[³H]alkyl-2-acetyl-sn-glycero-3-phosphocholinewas added to the platelets, the radioactivity was rapidly incorporatedinto the 1-alkyl-2-acyl-sn-glycero-3-phosphocholinefraction. These results indicate that1) continuous binding of PAF to itsreceptor is necessary for prolonged platelet aggregation, which may bemediated through an unknown signaling system for a long-term cellresponse rather than a transient signaling system, and2) most of the[³H]acetyl-PAF boundto platelets is metabolized extracellularly by ecto-type PAFacetylhydrolase, with the lyso-PAF generated being incorporated rapidlyinto the cells and converted to1-alkyl-2-acyl-sn-glycero-3-phosphocholine.

     

Phospholipases of Leptospira. I. Presence of phospholipase A1 and lysophospholipase in Leptospira biflexa

The hydrolysis of phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylcholine, and trioleoylglycerol by Leptospira biflexa strain Urawa was studied in vitro. Phospholipase A₁ was identified by the formation of ³²P- and ¹⁴C-labeled lyso-derivatives from ³²P-phosphatidylcholine, ³²P-phosphatidylethanolamine, or 1-acyl-2-[1-¹⁴C]oleoyl-sn-glycero-3-phosphorylcholine. Phospholipase A₁ activity was independent of lipase in the microorganism since ¹⁴C-labeled trioleoylglycerol was scarcely attacked under the same conditions in which the phospholipids were hydrolyzed. Lysophospholipase activity was also demonstrated using ³²P- and non-labeled lysophosphatidylcholine. The activity of phospholipase A₁ was found in a broad range of pH but no optimal pH was determined. The pH optimum of lysophospholipase was 8.0. Both enzymes were labile to heat. Phospholipase C activity, however, could not be detected because no radioactive di- and monoacylglycerol was found in the experiment with 1-acyl-2-[1-¹⁴C]-oleoyl-sn-glycero-3-phosphorylcholine as the substrate. It was inferred that phosphatidylethanolamine, which was the major component of phospholipids in leptospirae, was hydrolyzed serially by phospholipase A (A₁ and/or A₂?) and lysophospholipase to glycerophosphorylethanolamine via 2-acyl-type-lyso-derivative as one metabolic pathway of the substrate.     

Bradykinin-stimulated release of [H]arachidonic acid from phospholipids of HSDM1C1 cells: Comparison of diacyl phospholipids and plasmalogens as sources of prostaglandin precursors

Ethanolamine plasmalogens (1-alk-1′-enyl-2-acyl-sn-glycero-3-phosphoethanolamines) of many tissues contain high levels of arachidonate at their 2-position, and in certain tissues have been implicated as possible donors of arachidonate required in the synthesis of prostaglandins and thromboxanes. In the present study, [³H]arachidonate-labeled phospholipids of HSDM₁C₁ cells, a cell line derived from a mouse fibrosarcoma, were examined to determine the donor of the arachidonic acid released upon bradykinin stimulation of the synthesis of PGE₂. HSDM₁C₁ cells labeled with [³H]arachidonic acid for 24 hr in serum-free medium were used in most of the experiments and had the following distribution of label among the cellular lipids; phosphatidylcholine (33%), phosphatidylinositol (20%), diacyl-sn-glycero-3-phosphoethanolamine (15%), ethanolamine plasmalogen (15%), and less polar lipids (16%). Bradykinin treatment stimulated a rapid hydrolysis of [³H]arachidonate from the cellular lipids and conversion of the released acid to PGE₂, which was secreted into the medium. The label was released predominantly from phosphatidylinositol and possibly from phosphatidylcholine with no detectable change in the labeling of diacyl- or 1-alk-1′-enyl-2-acyl-sn-glycero-3-phosphoethanolamine. The ethanolamine plasmalogens, therefore, do not appear to be involved in the stimulated release of arachidonate in the HSDM₁C₁ cells. Indomethacin blocked the bradykinin-stimulated synthesis of PGE₂ and to a lesser degree inhibited the release of [³H]-arachidonate from the cellular lipids into the medium.     

Synthesis of carbamyl and ether analogs of phosphatidylcholines

A complete synthesis of 1-O-hexadecyl-2-O-N-(heptadec-8-cis-enyl)carbamyl-sn-glycero-3-Phosphocholine, a novel analog of phosphatidylcholine, has been described. Each step is simple to perform and gives the desired products in high yield. Also, some of the intermediates formed during the synthesis have been efficiently utilized to prepare 1-O-hexadecyl-2-O-oleyl-sn-glycero-3-phosphocholine, 1-O-hexadecyl-2-oleoyl-sn-glycero-3-phosphochloine and 3-O-hexadecyl-2-oeloyl-sn-glycero-1-phosphocholine. These phosphatidylcholine (PC) analogs are useful for studying the possible role of phospholipases in the capture and lyses of liposomes in vivo.     

Angiotensin II induces phosphatidic acid formation in neonatal rat cardiac fibroblasts: Evaluation of the roles of phospholipases C and D

Phosphatidic acid has been proposed to contribute to the mitogenic actions of various growth factors. In³²P-labeled neonatal rat cardiac fibroblasts, 100 nM [Sar¹]angiotensin II was shown to rapidly induce formation of³²P-phosphatidic acid. Levels peaked at 5 min (1.5-fold above control), but were partially sustained over 2 h. Phospholipase D contributed in part to phosphatidic acid formation, as³²P- or³H-phosphatidylethanol was produced when cells labeled with [³²P]H₃PO₄ or 1-O-[1,2-³H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine were stimulated in the presence of 1% ethanol. [Sar¹]angiotensin II-induced phospholipase D activity was transient and mainly mediated through protein kinase C (PKC), since PKC downregulation reduced phosphatidylethanol formation by 68%. Residual activity may have been due to increased intracellular Ca²⁺, as ionomycin also activated phospholipase D in PKC-depleted cells. Phospholipase D did not fully account for [Sar¹]angiotensin II-induced phosphatidic acid: 1) compared to PMA, a potent activator of phospholipase D, [Sar¹]angiotensin II produced more phosphatidic acid relative to phosphatidylethanol, and 2) PKC downregulation did not affect [Sar¹]angiotensin II-induced phosphatidic acid formation. The diacylglycerol kinase inhibitor R59949 depressed [Sar¹]angiotensin II-induced phosphatidic acid formation by only 21%, indicating that activation of a phospholipase C and diacylglycerol kinase also can not account for the bulk of phosphatidic acid. Thus, additional pathways not involving phospholipases C and D, such asde novo synthesis, may contribute to [Sar¹]angiotensin II-induced phosphatidic acid in these cells. Finally, as previously shown for [Sar¹]angiotensin II, phosphatidic acid stimulated mitogen activated protein (MAP) kinase activity. These results suggest that phosphatidic acid may function as an intracellular second messenger of angiotensin II in cardiac fibroblasts and may contribute to the mitogenic action of this hormone on these cells. (Mol Cell Biochem141: 135–143, 1994)Abbreviations DAG diacylglycerol - DMSO dimethyl sulfoxide - lysoPC 1-O-hexadecyl-2-lyso-sn-glycero-3-phosphocholine - NRCF newborn rat cardiac fibroblasts - PA phosphatidic acid - PAPase phosphatidic acid phosphohydrolase - PC phosphatidylcholine - PEt phosphatidylethanol - PI phosphatidylinositol - PL (labeled) phospholipids - PLC phospholipase C - PLD phospholipase D Drs. G. W. Booz and M. M. Taher contributed equally to the work described here.     

Oxidatively modified fatty acyl chain determines physicochemical properties of aggregates of oxidized phospholipids

In vivo oxidation of glycerophospholipid generates a variety of products including truncated oxidized phospholipids (tOx-PLs). The fatty acyl chains at the sn-2 position of tOx-PLs are shorter in length than the parent non-oxidized phospholipids and contain a polar functional group(s) at the end. The effect of oxidatively modified sn-2 fatty acyl chain on the physicochemical properties of tOx-PLs aggregates has not been addressed in detail, although there are few reports that modified fatty acyl chain primarily determines the biological activities of tOx-PLs. In this study we have compared the properties of four closely related tOx-PLs which differ only in the type of modified fatty acyl chain present at the sn-2 position: 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), 1-palmitoyl-2-(9′-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC), 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC), and 1-palmitoyl-2-(5′-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC). Aggregates of individual tOx-PL in aqueous solution were characterized by fluorescence spectroscopy, size exclusion chromatography, native polyacrylamide and agarose gel electrophoresis. The data suggest that aggregates of four closely related tOx-PLs form micelle-like particles of considerably different properties. Our result provides first direct evidence that because of the specific chemical composition of the sn-2 fatty acyl chain aggregates of particular tOx-PL possess a distinctive set of physicochemical properties.     

1-O-Alkyl-2-acyl-sn-glycero-3-phosphocholine: A novel source of arachidonic acid in neutrophils stimulated by the calcium ionophore A23187

Rabbit peritoneal neutrophils incorporated [¹⁴C]arachidonic acid into seven molecular species of choline-containing phosphoglycerides. These 2-[¹⁴C]arachidonoyl species differed with respect to the alkyl ether or acyl residue bound at the $\text{sn}$-1 position; four of the seven were ether-linked. Stimulation with calcium ionophore A23187 induced a proportionate release of arachidonate from all seven molecular species: 40% of the released arachidonate came from alkyl ether species. Thus, 1-$\text{O}$-alkyl-2-arachidonoyl-$\text{sn}$-glycero-3-phosphocholine (GPC) is a significant source of metabolizable arachidonic acid. Since 1-$\text{O}$-alkyl-2-lyso-GPC is the metabolic precussor of platelet activating factor, these results further interrelate pathways forming arachidonate metabolites and platelet activating factor; they also supply a rationale for the observation that both classes of stimuli form concomitantly during cell activation.     

Secondary structure and backbone dynamics of Escherichia coli diacylglycerol kinase,as revealed by site-directed solid-state 13C NMR

To gain insight into secondary structure and backbone dynamics, we have recorded ¹³C NMR spectra of [3-¹³C]Ala-, [1-¹³C]Val-labeled Escherichia coli diacylglycerol kinase (DGK), using cross-polarization-magic angle spinning (CP-MAS) and single-pulse excitation with dipolar decoupled-magic angle spinning (DD-MAS) methods. DGK was either solubilized in n-decyl-β-maltoside (DM) micelle or integrated into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers. Surprisingly, the ¹³C NMR spectra were broadened to yield rather featureless peaks at physiological temperatures, both in DM solution or lipid bilayers at liquid crystalline phase, due to interference of motional frequencies of DGK with frequencies of magic angle spinning (MAS) or proton decoupling (10⁴ or 10⁵ Hz, respectively). In gel phase lipids, however, up to six distinct ¹³C NMR peaks were well-resolved due to lowered fluctuation frequencies (<10⁵ Hz) for the transmembrane region, the amphipathic α-helices and loops. While DGK can be tightly packed in gel phase lipids, DGK is less tightly packed at physiological temperatures, where it becomes more mobile. The fact that the enzymatic activity is low under conditions where motion is restricted and high when conformational fluctuations can occur suggests that acquisition of low frequency backbone motions, on the microsecond to millisecond time scale, may facilitate the efficient enzymatic activity of DGK.     

Acyltransferases and the biosynthesis of pulmonary surfactant lipid in adenoma alveolar type II cells.

Acyltransferases are present in microsomes from alveolar type II cell adenomas (produced by urethan injections) that transfer palmitic acid in the presence of CoA, ATP, and Mg⁺⁺ to $\text{sn}$-glycerol-3-P to form phosphatidic acid, to dihydroxyacetone-P to form acyldihydroxyacetone-P, and to 1-acyl-$\text{sn}$-glycero-3-phosphocholine to form 3-$\text{sn}$-phosphatidylcholine. The data clearly demonstrate that the microsomal preparations can catalyze significant incorporation of palmitic acid into the 2-position of the disaturated species of 3-sn-phosphatidylcholine independently of phosphatidic acid formation as evidenced by the fact that $\text{sn}$-glycerol-3-P and calcium ions (which inhibit choline phosphotransferase) did not influence the incorporation of palmitic acid into the main surfactant lipid. Thus, a deacylation-acylation reaction involving 2-lysophosphatidylcholine appears to be an important pathway for the synthesis of surfactant lipid in alveolar type II cells; the control of acyl specificity at the 2-position is determined by the relative concentrations of the coparticipating substrates, l-palmitoyl-$\text{sn}$-glycero-3-phosphocholine and palmitoyl-CoA.     

Structure and Dynamics of the Myristoyl Lipid Modification of Src Peptides Determined by H Solid-State NMR Spectroscopy

Lipid modifications of proteins are widespread in nature and play an important role in numerous biological processes. The nonreceptor tyrosine kinase Src is equipped with an N-terminal myristoyl chain and a cluster of basic amino acids for the stable membrane association of the protein. We used ²H NMR spectroscopy to investigate the structure and dynamics of the myristoyl chain of myr-Src(2-19), and compare them with the hydrocarbon chains of the surrounding phospholipids in bilayers of varying surface potentials and chain lengths. The myristoyl chain of Src was well inserted in all bilayers investigated. In zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine membranes, the myristoyl chain of Src was significantly longer and appears “stiffer” than the phospholipid chains. This can be explained by an equilibrium between the attraction attributable to the insertion of the myristoyl chain and the Born repulsion. In a 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-[phospho-L-serine] membrane, where attractive electrostatic interactions come into play, the differences between the peptide and the phospholipid chain lengths were attenuated, and the molecular dynamics of all lipid chains were similar. In a much thicker 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dipalmitoyl-sn-glycero-3-[phospho-L-serine]/cholesterol membrane, the length of the myristoyl chain of Src was elongated nearly to its maximum, and the order parameters of the Src chain were comparable to those of the surrounding membrane.     

Metabolism of long-chain alcohols in cell suspension cultures of soya and rape

Heterotrophic cell suspension cultures of soya (Glycine max) and photomixotrophic cell suspension cultures of rape (Brassica napus) were incubated with cis-9-[1-¹⁴C]octadecenol for 3–48 h. It was found that under aerobic conditions large proportions of the alcohol are oxidized to oleic acid, which is incorporated predominantly into phospholipids, whereas up to 30% of the substrate is esterified to wax esters. This is true for both the heterotrophic and the photomixotrophic cell suspension cultures, but the metabolic rates are much higher in the latter. Under anaerobic conditions only small proportions of the radioactively labeled alcohol are oxidized to oleic acid, whereas a major portion of the alcohol is esterified to wax esters both in heterotrophic and photomixotrophic cultures. Incubations of homogenates of photomixotrophic rape cells with labeled cis-9-octadecenol showed that pH 6 is optimum for the formation of wax esters. This monounsaturated alcohol is preferred as a substrate over saturated longchain alcohols, whereas short-chain alcohols, cholesterol, and glycerol are not acylated. Incubations of an enzyme concentrate from a homogenate of rape cells with unlabeled cis-9-octadecenol and [1-¹⁴C]oleic acid, or [1-¹⁴C]stearoyl-CoA, or di[1-¹⁴C]palmitoyl-sn-glycero-3-phosphocholine showed that acylation of the longchain alcohol proceeds predominantly through acyl-CoA. Direct esterification of the alcohol with fatty acid as well as acyl transfer from diacylglycerophosphocholine could be demonstrated to occur to a much smaller extent.