Exogenous Phosphatidylethanolamine Research Articles

Phosphatidylethanolamine Improves Postnatal Growth Retardation by Regulating Mucus Secretion of Intestinal Goblet Cells in Piglets.

Phosphatidylethanolamine (PE), a multifunctional phospholipid, is necessary for neonate development. This study aimed to explore the impact of the regulation of exogenous PE on postnatal growth retardation (PGR) by improving intestinal barrier function. Thirty-two neonatal pigs were divided into four groups according to their body weight (BW 2.79 ± 0.50 kg or 1.88 ± 0.40 kg) at 7 days old, CON-NBW, PE-NBW, CON-PGR, and PE-PGR. PE was supplemented to NBW piglets and PGR piglets during lactation and post-weaning periods. Compared with the NBW piglets, the growth performance of PGR piglets was lower, while PE improved the poor growth performance. PGR piglets showed injured intestinal morphology, as evidenced by the reduced ratio of villus height to crypt depth (VH/CD) and goblet cell numbers in the jejunum and ileum. PE recovered the intestinal barrier injury by increasing VH/CD and goblet cell numbers. The decreased MUC2 mRNA and protein expressions were observed in the small intestine of PGR piglets, and PE remarkably increased the expression of MUC2. Mechanistically, PE increased the goblet cell differentiation promoting gene spdef mRNA levels and reduced the mRNA expressions involved in endoplasmic reticulum stress in the jejunal and ileal mucosa of PGR piglets. Overall, we found that PE alleviated growth retardation by regulating intestinal health and generalized its application in neonates.

Phosphatidylethanolamine alleviates OX-LDL-induced macrophage inflammation by upregulating autophagy and inhibiting NLRP1 inflammasome activation

Oxidized low-density lipoprotein (OX-LDL)-induced inflammation and autophagy dysregulation are important events in the progression of atherosclerosis. Phosphatidylethanolamine (PE), a multifunctional phospholipid that is enriched in cells, has been proven to be directly involved in autophagy which is closely associated with inflammation. However, whether PE can influence OX-LDL-induced autophagy dysregulation and inflammation has not been reported. In the present study, we revealed that OX-LDL significantly induced macrophage inflammation through the CD36-NLRP1-caspase-1 signaling pathway in fish. Meanwhile, cellular PE levels were significantly decreased in response to OX-LDL induction. Based on the relationship between PE and autophagy, we then examined the effect of PE supplementation on OX-LDL-mediated autophagy impairment and inflammation induction in macrophages. As expected, exogenous PE restored impaired autophagy and alleviated inflammation in OX-LDL-stimulated cells. Notably, autophagy inhibitors reversed the inhibitory effect of PE on OX-LDL-induced maturation of IL-1β, indicating that the regulation of PE on OX-LDL-induced inflammation is dependent on autophagy. Furthermore, the positive effect of PE on OX-LDL-induced inflammation was relatively conserved in mouse and fish macrophages. In conclusion, we elucidated the role of the CD36-NLRP1-caspase-1 signaling pathway in OX-LDL-induced inflammation in fish and revealed for the first time that altering PE abundance in OX-LDL-treated cells could alleviate inflammasome-mediated inflammation by inducing autophagy. Given the relationship between OX-LDL-induced inflammation and atherosclerosis, this study prompts that the use of PE-rich foods promises to be a new strategy for atherosclerosis treatment in vertebrates.

Mathermycin, an anti-cancer molecule that targets cell surface phospholipids

Mathermycin, a lantipeptide isolated from marine actinomycete Marinactinospora thermotolerans, is an antibiotic that has been shown to disrupt bacterial plasma membrane. We now provide evidences that mathermycin can also disrupt cancer, but not normal, cell plasma membranes through targeting phosphatidylethanolamine (PE), which is located only in the inner leaflet of the plasma membrane in normal cells but in both the inner and outer leaflets of the membrane in tumor cells. Our data shows that mathermycin inhibits the metabolic activity and induces mainly necrotic death of all cancer cell lines with EC50 between 4.2 and 16.9 μM, while normal cell lines have EC50 between 113 and 129 μM. The cytotoxicity of mathermycin could be inhibited by exogenous PE, but not phosphoserine and phosphocholine. The formation of mathermycin-PE complexes was confirmed by in silico analysis, HPLC and MS spectrometer. Furthermore, mathermycin exhibited similar cytotoxicity toward cancer and multidrug resistant cancer cells, which could be due to its ability to inhibit mitochondrial function, as shown by our data from the Seahorse™ metabolic analyzer. This study demonstrates that mathermycin is a potentially effective class of anti-tumor chemotherapeutics that do not easily develop resistance due to a mechanism of action targeting PE.

Import of phosphatidylserine to and export of phosphatidylethanolamine molecular species from mitochondria

Heavy isotope-labeled ethanolamine and serine as well as exogenous PE and PS species were used to study trafficking of phosphatidylethanolamine (PE) and -serine (PS) molecular species between the endoplasmic reticulum (ER) and mitochondria in HeLa cells. Import of both endogenous and exogenous PS to IMM was a relatively slow process (T1/2=several hours), but depended on the acyl chains. In particular, the 38:4 and 38:5 species were imported more efficiently compared to the other PS species. Knock-down of Mitofusin 2 or Mitostatin had no detectable effect on PS import to mitochondria, suggesting that the ER–mitochondria contacts regulated by these proteins are not essential. Knock-down of PS synthase 1 inhibited PS decarboxylation, suggesting that import of PS to mitochondria is coupled to its synthesis. Also the export of PE from IMM to microsomes is a relatively slow process, but again depends markedly on the acyl chain structure. Most notably, the polyunsaturated 38:4 and 38:5 PE species were less efficiently exported, which together with rapid import of the PS precursors most probably explains their enrichment in IMM. PE synthesized via the CDP-ethanolamine was also imported to IMM, but most of the PE in this membrane derives from imported PS. In contrast to PS, all PC species made in Golgi/ER translocated similarly and rapidly to IMM. In conclusion, selective translocation of PS species and PS-derived PE species between ER and mitochondria plays a major role in phospholipid homeostasis of these organelles.

Isolation and characterization of a mutant defective in utilization of exogenous phosphatidylethanolamine in Saccharomyces cerevisiae

Isolation and characterization of a mutant defective in utilization of exogenous phosphatidylethanolamine in Saccharomyces cerevisiae

The linoleic acid derivative DCP-LA selectively activates PKC-ɛ, possibly binding to the phosphatidylserine binding site

This study examined the effect of 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), a newly synthesized linoleic acid derivative with cyclopropane rings instead of cis-double bonds, on protein kinase C (PKC) activity. In the in situ PKC assay with reverse-phase high-performance liquid chromatography, DCP-LA significantly activated PKC in PC-12 cells in a concentration-dependent (10 nM-100 microM) manner, with the maximal effect at 100 nM, and the DCP-LA effect was blocked by GF109203X, a PKC inhibitor, or a selective inhibitor peptide of the novel PKC isozyme PKC-epsilon. Furthermore, DCP-LA activated PKC in HEK-293 cells that was inhibited by the small, interfering RNA against PKC-epsilon. In the cell-free PKC assay, of the nine isozymes examined here, DCP-LA most strongly activated PKC-epsilon, with >7-fold potency over other PKC isozymes, in the absence of dioleoyl-phosphatidylserine and 1,2-dioleoyl-sn-glycerol; instead, the DCP-LA action was inhibited by dioleoyl-phosphatidylserine. DCP-LA also activated PKC-gamma, a conventional PKC, but to a much lesser extent compared with that for PKC-epsilon, by a mechanism distinct from PKC-epsilon activation. Thus, DCP-LA serves as a selective activator of PKC-epsilon, possibly by binding to the phosphatidylserine binding site on PKC-epsilon. These results may provide fresh insight into lipid signaling in PKC activation.

Enteropathogenic Escherichia coli Infection Triggers Host Phospholipid Metabolism Perturbations

Enteropathogenic Escherichia coli (EPEC) specifically recognizes phosphatidylethanolamine (PE) on the outer leaflet of host epithelial cells. EPEC also induces apoptosis in epithelial cells, which results in increased levels of outer leaflet PE and increased bacterial binding. Consequently, it is of interest to investigate whether EPEC infection perturbs host cell phospholipid metabolism and whether the changes play a role in the apoptotic signaling. Our findings indicate that EPEC infection results in a significant increase in the epithelial cell PE level and a corresponding decrease in the phosphatidylcholine (PC) level. PE synthesis via both the de novo pathway and the serine decarboxylation pathway was enhanced, and de novo synthesis of phosphatidylcholine via CDP-choline was reduced. The changes were transitory, and the maximum change was noted after 4 to 5 h of infection. Addition of exogenous PC or CDP-choline to epithelial cells prior to infection abrogated EPEC-induced apoptosis, suggesting that EPEC infection inhibits the CTP-phosphocholine cytidylyltransferase step in PC synthesis, which is reportedly inhibited during nonmicrobially induced apoptosis. On the other hand, incorporation of exogenous PE by the host cells enhanced EPEC-induced apoptosis and necrosis without increasing bacterial adhesion. This is the first report that pathogen-induced apoptosis is associated with significant changes in PE and PC metabolism, and the results suggest that EPEC adhesion to a host membrane phospholipid plays a role in disruption of host phospholipid metabolism.

Ca2+-induced Phosphoethanolamine Transfer to the Outer 3-Deoxy-d-manno-octulosonic Acid Moiety of Escherichia coli Lipopolysaccharide: A NOVEL MEMBRANE ENZYME DEPENDENT UPON PHOSPHATIDYLETHANOLAMINE

Certain strains of Escherichia coli and Salmonella contain lipopolysaccharide (LPS) modified with a phosphoethanolamine (pEtN) group at position 7 of the outer 3-deoxy-d-manno-octulosonic acid (Kdo) residue. Using the heptose-deficient E. coli mutant WBB06 (Brabetz, W., Muller-Loennies, S., Holst, O., and Brade, H. (1997) Eur. J. Biochem. 247, 716-724), we now demonstrate that the critical parameter determining the presence or absence of pEtN is the concentration of CaCl(2) in the medium. As judged by mass spectrometry, half the LPS in WBB06, grown on nutrient broth with 5 mm CaCl(2), is derivatized with a pEtN group, whereas LPS from WBB06 grown without supplemental CaCl(2) is not. Membranes from E. coli WBB06 or wild-type W3110 grown on 5-50 mm CaCl(2) contain a novel pEtN transferase that uses the precursor Kdo(2)-[4'-(32)P]lipid IV(A) as an acceptor. Transferase is not present in membranes of E. coli grown with 5 mm MgCl(2), BaCl(2), or ZnCl(2). Hydrolysis of the in vitro reaction product, pEtN-Kdo(2)-[4'-(32)P]lipid IV(A), at pH 4.5 shows that the pEtN substituent is located on the outer Kdo moiety. Membranes from an E. coli pss knockout mutant grown on 50 mm CaCl(2), which lack phosphatidylethanolamine, do not contain measurable transferase activity unless exogenous phosphatidylethanolamine is added back to the assay system. The induction of the pEtN transferase by 5-50 mm CaCl(2) suggests possible role(s) in establishing transformation competence or resisting environmental stress, and represents the first example of a regulated covalent modification of the inner core of E. coli LPS.

Myxococcus xanthus fibril appendages are essential for excitation by a phospholipid attractant.

Isolated (A-motile) Myxococcus xanthus cells glide over solid surfaces and display excitation, a suppression of direction reversals, when presented with phosphatidylethanolamine (PE) purified from its own membranes or synthetic dilauroyl PE and dioleoyl PE. Although the mechanism of PE signal transduction is unknown, we hypothesized that M. xanthus might use surface-associated factors to detect exogenous PE to prevent endogenous lipids from self-stimulating the sensory system. Peritrichous protein and polysaccharide appendages called fibrils were correlated with dilauroyl PE excitation. Wild-type cells treated with Congo red, an inhibitor of fibril assembly, and mutants defective in fibril biosynthesis showed an elevated reversal period, which suggested that fibrils regulate the gliding motor. Furthermore, the loss of fibrils resulted in loss of excitation to dilauroyl PE but not dioleoyl PE. Restoration of fibril production to these mutants restored the dilauroyl PE response. In addition, the dif cytoplasmic signal transduction system and starvation conditions were required for dilauroyl PE excitation. The chemically specific nature of the response and the dependence on the dif system suggests that fibrils define a novel sensory organelle whose evolution may have been necessary to prevent autostimulation by endogenous membrane lipids. Because the hydrophobic nature of dilauroyl PE would be inaccessible to periplasmic chemosensors, we suggest that fibrils act as extracellular signal transducers to probe surfaces for insoluble chemical signals.

Activity of phosphatidylethanolamine-N-methyltransferase in brain affected by Alzheimers disease

To determine whether phospholipid abnormality in Alzheimers disease is associated with modification of phosphatidylethanolamine- N-methyltransferase, the activity of the enzyme was analysed in the frontal and occipital cortex of the brain from patients with Alzheimers disease and from aged-matched control. The optimum pH for phosphatidylethanolamine- N-methyltransferase in human brain was 9.0. The enzyme activity was stimulated by detergent TWEEN 20 but inhibited by Triton X-100. Neither magnesium dependence nor chemical methylation was found. A decrease in activity of phosphatidylethanolamine- N-methyltransferase was observed in the frontal cortex of brain affected with Alzheimers disease. The addition of exogenous phosphatidylethanolamine resulted in no modification in the methylation rate as compared with that of endogenous PE. The addition of phosphatidyl- N-monomethylethanolamine and phosphatidyl- N, N-dimethylethanolamine resulted in significantly increased rates of methylation in brain tissues. However, the increased rate of phosphatidylethanolamine- N-methyltransferase activity stimulated by exogenous phospholipids was lower in the frontal cortex of brains with Alzheimers disease when compared to the normals and there was no difference in the occipital cortex between Alzheimers disease and the control. It is plausible that the decreased activity of phosphatidylethanolamine- N-methyltransferase and its low compensating ability could relate to the modification of phosphatidylcholine in brain tissues from Alzheimers disease patients.

Genetic Evidence That Phosphatidylserine Synthase II Catalyzes the Conversion of Phosphatidylethanolamine to Phosphatidylserine in Chinese Hamster Ovary Cells

Phosphatidylserine (PS) in mammalian cells is synthesized through the exchange of free L-serine with the base moiety of phosphatidylcholine or phosphatidylethanolamine (PE). The serine base exchange in Chinese hamster ovary (CHO) cells is catalyzed by at least two enzymes, PS synthase (PSS) I and II. A PSS I-lacking mutant of CHO-K1 cells, PSA-3, which exhibits approximately 2-fold lower serine base exchange activity than CHO-K1, is defective in the conversion of phosphatidylcholine to PS but has the ability to convert PE to PS. The PSA-3 mutant requires exogenous PS or PE for cell growth. In the present study, from PSA-3 mutant cells, we isolated a mutant, named PSB-2, with a further decrease in the serine base exchange activity. The activity in the homogenate of PSB-2 mutant cells was approximately 10% that of PSA-3 mutant cells and approximately 5% that of CHO-K1 cells. The PSB-2 mutant exhibited an approximately 80% reduction in the PSS II mRNA level relative to that in PSA-3 mutant and CHO-K1 cells. These results showed that the PSB-2 mutant is defective in PSS II. Like the PSA-3 mutant, the PSB-2 mutant grew well in medium supplemented with PS. However, in the medium supplemented with PE, the PSB-2 mutant was incapable of growth, in contrast to the PSA-3 mutant. In the medium with exogenous PE, the PSB-2 mutant was defective in PS biosynthesis, whereas the PSA-3 mutant synthesized a normal amount of PS. A metabolic labeling experiment with exogenous [32P]PE revealed that the PSB-2 mutant was defective in the conversion of exogenous PE to PS. This defect and the growth and PS biosynthetic defects of the PSB-2 mutant cultivated with exogenous PE were complemented by the PSS II cDNA. In addition, the cDNA of the other PS synthase, PSS I, was shown not to complement the defect in the conversion of exogenous PE to PS of the PSB-2 mutant, implying that PSS I negligibly contributes to the conversion of PE to PS in CHO-K1 cells. These results indicated that PSS II is critical for the growth and PS biosynthesis of PSA-3 mutant cells cultivated with exogenous PE and suggested that most of the PS formation from PE in CHO-K1 cells is catalyzed by PSS II.

Activation of Murine Lymphocytes by Exogenous Phosphatidylethanolamine- and Phosphatidylcholine-Specific Phospholipase C

Enzymes of the protein kinase C (PKC) family have a decisive role in the activation process of T cells. Under physiological conditions. PKC undergoes activation by diacylglycerol (DAG) which is transiently produced following activation of an antigen receptor-coupled phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phospholipase C (PLC) and hydrolysis of PIP2. In vitro activation of distinct PKC isoenzymes is differentially regulated by various synthetic DAGs and membrane phospholipids. We tested whether exogenous PLC obtained from Bacillus cereus can affect PKC-dependent functions in mouse lymphocytes. The results indicated that B. cereus PLC, which preferentially hydrolyzes phosphatidylethanolamine (PE) and phosphatidylcholine (PC), induces the expression of functional cell surface IL2 receptors and that, in the presence of exogenous IL2, it leads to cell proliferation. The PE/PC-PLC reversed the inhibitory effect of PC on enzymatic activity of T cell-derived PKC in vitro. Furthermore, it augmented the activity of PKC over the background level suggesting that the PC-derived DAG may function as a positive regulator of T cell-specific PKC isoenzymes.

Phospholipid methylation in brain membrane preparations: Kinetic mechanism

The methylation reactions which convert phosphatidylethanolamine (PE) to phosphatidylcholine (PC) have been studied kinetically using exogenously added intermediates and crude membrane preparations from brain. The addition of exogenous PE resulted in no change in the methylation rates compared to that of endogenous PE. The addition of the two intermediates, monomethylphosphatidylethanolamine (PMME) and dimethylphosphatidylethanolamine (PDME), resulted in significantly increased rates of methylation and allowed the kinetic analysis of these latter two methylation reactions. The mechanism for this enzyme appears to be similar to human RBC (Reitz et al. (1989) J. Biol. Chem. 264, 8097–8106) which was a rapid-equilibrium random Bi-Bi sequential mechanism. There were some slight differences between the brain enzyme and that from the RBC, but there is little reason to suggest a fundamentally different mechanism. It is more likely that the differences may relate to an additional dead-end complex for the enzyme from brain such that saturation with AdoMet cannot eliminate AdoHcy inhibition. The K M values for the two phospholipid substrates were 41–44 μM and 39 μM for the methylation of PMME and PDME, respectively. The K M for S-adenosylmethionine (AdoMet) was 7–9 μM with PMME and 4 μM with PDME as the other substrates. The K i(lipid) varied from 54 μM with PMME to 225 μM with PDME, and the K i(AdoMet) was 11 μM with PMME and 21 μM with PDME. The product from the use of AdoMet, S-adenosylhomocysteine (AdoHcy), was shown to be a noncompetitive inhibitor of both lipid substrates as well as AdoMet. The methylation of PMME was somewhat higher in cerebellum and brain stem compared to cortex and striatum, but the methylation of PDME was similar in cerebellum, brain stem and cortex.

Effect of polyunsaturated fatty acids and phospholipids on “3H”‐vitamin E incorporation into pulmonary artery endothelial cell membranes

Vitamin E, a dietary antioxidant, is presumed to be incorporated into the lipid bilayer of biological membranes to an extent proportional to the amount of polyunsaturated fatty acids or phospholipids in the membrane. In the present study we evaluated the distribution of incorporated polyunsaturated fatty acids (PUFA) and phosphatidylethanolamine (PE) in various membranes of pulmonary artery endothelial cells. We also studied whether incorporation of PUFA or PE is responsible for increased incorporation of [3H]-vitamin E into the membranes of these cells. Following a 24-hr incubation with linoleic acid (18:2), 18:2 was increased by 6.9-, 9.2-, and 13.2-fold in plasma, mitochondrial, and microsomal membranes, respectively. Incorporation of 18:2 caused significant increases in the unsaturation indexes of mitochondrial and microsomal polyunsaturated fatty acyl chains (P less than .01 versus control in both membranes). Incubation with arachidonic acid (20:4) for 24 hr resulted in 1.5-, 2.3-, and 2.4-fold increases in 20:4 in plasma, mitochondrial, and microsomal membranes, respectively. The unsaturation indexes of polyunsaturated fatty acyl chains of mitochondrial and microsomal membranes also increased (P less than .01 versus control in both membranes). Although incubations with 18:2 or 20:4 resulted in several-fold increases in membrane 18:2 or 20:4 fatty acids, incorporation of [3H]-vitamin E into these membranes was similar to that in controls. Following a 24-hr incubation with PE, membrane PE content was significantly increased, and [3H]-vitamin E incorporation was also increased to a comparable degree, i.e., plasma membrane greater than mitochondria greater than microsomes. Endogenous vitamin E content of the cells was not altered because of increased incorporation of PE and [3H]-vitamin E. When [3H]-vitamin E was incorporated into lipid vesicles prepared from the total lipid extracts of endothelial cells and varying amounts of exogenous PE, vitamin E content was directly related to PE content. These results demonstrate that PUFA and PE distribute in all pulmonary artery endothelial cell membranes. However, only increases in PE were associated with increased incorporation of [3H]-vitamin E in membranes of these cells.

Phosphatidylethanolamine activation of the purified and delipidated cytochrome-c4:o oxidase from Azotobacter vinelandii

The effects of exogenous phosphatidylethanolamine (PE) on the activity of a purified and a delipidated cytochrome oxidase from Azotobacter vinelandii were studied. The Vmax values of N,N,N′,N′-tetramethyl-p-phenylenediamine oxidation by the cytochrome oxidase increased linearly as a direct function of the amount of exogenously added PE. Some PE remained bound to the oxidase, even after extensive delipidation. Results indicated PE might serve a vital function in maintaining the enzymic activity of this oxidase in A. vinelandii.

Characterization of phospholipase A2 from rabbit lung microsomes.

A phospholipase A2 activity associated with the microsomal fraction of rabbit lung homogenates was studied. The enzyme showed specificity for the sn-2 ester bond of phosphatidylcholine, had an alkaline pH optimum and required Ca2+ for activity. Other divalent cations were unable to support hydrolysis. In the absence of detergents, exogenous phosphatidylethanolamine was deacylated at a rate sevenfold higher than phosphatidylcholine. The activity toward both substrates could be enhanced by sodium deoxycholate or, more effectively, by sodium taurodeoxycholate. Phosphatidylethanolamine required higher detergent/phospholipid molar ratios than phosphatidylcholine. Under these conditions, the preference for the former substrate over the latter was nearly abolished. The zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) and the nonionic detergent Triton X-100 were either ineffective (phosphatidylcholine) or inhibitory (phosphatidylethanolamine). Addition of KCl produced opposite effects on the activity depending on the bile salt used to disperse the substrate. The phospholipase A2 activity was inhibited by p-bromophenacyl bromide but remained unaffected after treatment with diisopropylfluorophosphate or NaF. N-Ethylmaleimide, but not other thiol reagents, partially inhibited the activity.

Sphingomyelin and ceramide-phosphoethanolamine synthesis by microsomes and plasma membranes from rat liver and brain.

Pulse-chase experiments showed that phosphatidylethanolamine (PE) was the direct precursor for ceramide-phosphoethanolamine, a sphingomyelin analogue, in the same way as phosphatidylcholine was for sphingomyelin. Ceramide-phosphoethanolamine could be identified by incorporation of radioactivity from labeled PE, as well as by its stability in alkaline methanolysis and its ability to be methylated by S-adenosyl-methionine. Ceramide-phosphoethanolamine synthesis from labeled exogenous PE seemed to be independent of exogenous ceramide; it was proportional to the amount of incubated membrane, when taking into account the isotopic dilution of labeled precursor by endogenous PE. Sphingomyelin synthesis, which was demonstrated using natural PC as a substrate, was not possible using dipalmitoyl-PC. The formation of sphingomyelin and ceramide-phosphoethanolamine was demonstrated in microsomes and plasma membranes from rat brain and liver.

Phosphatidylserine biosynthesis in cultured Chinese hamster ovary cells. III. Genetic evidence for utilization of phosphatidylcholine and phosphatidylethanolamine as precursors.

In the preceding paper, we reported that Chinese hamster ovary (CHO) cells contain two different serine-exchange enzymes (I and II) which catalyze the base-exchange reaction of phospholipid(s) with serine and that a phosphatidylserine-requiring mutant (strain PSA-3) of CHO cells is defective in serine-exchange enzyme I and lacks the ability to synthesize phosphatidylserine (Kuge, O., Nishijima, M., and Akamatsu, Y. (1986) J. Biol. Chem. 261, 5790-5794). In this study, we examined precursor phospholipids for phosphatidylserine biosynthesis in CHO cells. When mutant PSA-3 and parent (CHO-K1) cells were cultured with [32P]phosphatidylcholine, phosphatidylserine in the parent accumulated radioactivity while that in the mutant was not labeled significantly. On the contrary, when cultured with [32P]phosphatidylethanolamine, the mutant incorporated the label into phosphatidylserine more efficiently than the parent. Furthermore, we found that mutant PSA-3 grew normally in growth medium supplemented with 30 microM phosphatidylethanolamine as well as phosphatidylserine and that the biosynthesis of phosphatidylserine in the mutant was biosynthesis of phosphatidylserine in the mutant was normal when cells were cultured in the presence of exogenous phosphatidylethanolamine. The simplest interpretation of these findings is that phosphatidylserine in CHO cells is biosynthesized through the following sequential reactions: phosphatidylcholine----phosphatidylserine----phosphatidylethanolamine--- - phosphatidylserine. The three reactions are catalyzed by serine-exchange enzyme I, phosphatidylserine decarboxylase, and serine-exchange enzyme II, respectively.

Intracellular phospholipase activities of Tetrahymena pyriformis.

Phospholipase activity was studied in the protozoan Tetrahymena pyriformis NT-1 by using exogenous phosphatidylethanolamine and phosphatidylcholine. Several phospholipase activities were found in Tetrahymena homogenates. They were distinguished with respect to pH optimum, activity dependence on Ca2+, substrate specificity and positional specificity. Ca2+-Dependent phospholipase activity had an optimal pH around 9 and gave rise to free fatty acid and lysophospholipid. This enzyme hydrolyzes phosphatidylethanolamine but not phosphatidylcholine. The alkaline phospholipase with A1 activity was located mainly in the surface membrane (pellicle fraction). The enzyme activity had a pH optimum ranging from 8 to 9, and required 2 mM CaCl2 for the maximal activity. All detergents tested inhibited the enzyme activity. Ca2+-Independent phospholipase activity had an optimal pH from 4 to 5 and gave rise to free fatty acid, lysophospholipid, diacylglycerol, and monoacylglycerol. We concluded that there are at least three phospholipase in Tetrahymena homogenates, i.e., alkaline phospholipase A and acidic phospholipases A and C.

Regulatory aspects of rat liver mitochondrial phospholipase A 2: Effects of calcium ions and calmodulin

A comparative study was made of the metal ion requirement of rat liver mitochondrial phospholipase A 2 in purified and membrane-associated forms. Membrane-bound enzyme was assayed using either exogenous or endogenous phosphatidylethanolamine. Although several divalent metal ions caused increased activity of the membrane-associated enzyme, only Ca 2+ and Sr 2+ activated the purified phospholipase A 2. The activity in the presence of Sr 2+ amounted to about 25% of that found with Ca 2+. When the Ca 2+ concentration was varied two activity plateaus were observed. The corresponding dissociation constants varied from 6 to 20 μ m Ca 2+ and from 1.4 to 12 m m Ca 2+ for the high- and low-affinity binding sites, respectively, depending on the assay conditions and whether purified or membrane-bound enzyme was used. A k Sr 2+ of 60 μ m was found for the high-affinity binding site. The effect of calmodulin and its antagonist trifluoperazine was also investigated using purified and membrane-associated enzyme. When membrane-bound enzyme was measured with exogenous phosphatidylethanolamine, small stimulations by calmodulin were found. However, these were not believed to indicate a specific role for calmodulin in the Ca 2+ dependency of the phospholipase A 2, since trifluoperazine did not lower the activity of the membrane-bound enzyme to levels below those found in the presence of Ca 2+ alone. Membrane-bound enzyme in its action toward endogenous phosphatidylethanolamine was neither stimulated by calmodulin nor inhibited by trifluoperazine. Purified enzyme was also not stimulated by calmodulin, while trifluoperazine caused small stimulations, presumably due to interactions at the substrate level. These results indicate that calmodulin involvement in phospholipase A 2 activation should not be generalized.