Phospholipid metabolism in cells in culture

The proportions of the different lecithin fractions have been determined in HeLa and KB tissue culture cells and Ehrlich Ascites tumor. 82.8% of the total phosphatidyl choline phosphorus is found in fraction 3 of HeLa cells. The major phosphatidyl cholines found in KB cells and Ehrlich Ascites tumor are in fractions 3 and 4 and representing 66.6% and 88.7% of the total phosphatidyl choline P, respectively. The incorporation of 1,2-¹⁴C-choline and 1,2-¹⁴C-ethanolamine into the various phosphatidyl choline fractions has been assayed to determine their biosynthesis in Ehrlich Ascites tumor. The incorporation of 1,2-¹⁴C-choline into fractions 3 and 4 is 100 times the 1,2-¹⁴C-ethanolamine. This evidence indicates that the methylation pathway of phosphatidyl choline synthesis is very low in HeLa, KB and Ehrlich Ascites cells. One of 13 papers presented at the symposium “Lipid Metabolism in Cells in Culture,” AOCS Meeting, Houston, May 1971. Part of a thesis submitted to the Graduate School of the University of North Dakota in partial fulfillment of the Degree of Master of Science.     

Phospholipid reactivation of plasmalogen metabolism

This report is concerned mainly with the properties of an enzyme from rat liver microsomes which hydrolyzes the alkenyl ether bond of 1-(1′-alk-1′-enyl)-glycero-3-phosphoryl-choline (alkenyl-GPC hydrolase). Destruction of the normal environment of the microsomes by treatment with phospholipases A or C caused inactivation of the alkenyl-GPC hydrolase, which was then partially reactivated by the addition of exogenous phospholipids. Both sphingomyelin and diacyl-GPC were efficient in restoring activity; diacyl-GPE was less effective; and monoacyl-GPC and monoacyl-GPE were ineffective. The presence of two long hydrocarbon chains in the lipid activator is apparently required for reactivation, suggesting that interaction of hydrophobic areas of the enzyme with the phospholipid is necessary for maximal activity. High concentrations of sucrose mimicked the effect of phospholipids, and because the sucrose and diacyl-GPC did not show an additive effect, they may reactivate the enzyme in a similar manner. Disrupting the enzyme's environment by freezing and thawing the preparation also resulted in a loss of enzymatic activity, which was restored by added exogenous phospholipids. The alkenyl-GPC hydrolase was inhibited by imidazole and some of its derivatives. Histidine and N-acetyl histidine did not inhibit the enzyme, presumably due to the presence of a negative charge on the carboxyl group rather than the steric bulk of that group, since histidine methyl ester did inhibit the enzyme. Kinetic evidence showed imidazole to be a competitive inhibitor. The enzymatic activity of imidazole-treated microsomes also increased following addition of exogenous phospholipids. Imidazole inhibition differed from the phospholipase A-inactivation in that it was partially reversed by KCl, but not by sucrose. Imidazole did not inhibit other microsomal enzymes tested, indicating that it is not a general inhibitor of membrane-associated enzymes.     

Phospholipid composition of cultured human endothelial cells

Detailed analyses of the phospholipid compositions of cultured human endothelial cells are reported here. No significant differences were found between the phospholipid compositions of cells from human artery, saphenous and umbilical vein. However, due to the small sample sizes, relatively large standard deviations for some of the phospholipid classes were observed. A representative composition of endothelial cells is: phosphatidylcholine 36.6%, choline plasmalogen 3.7%, phosphatidylethanolamine 10.2%, ethanolamine plasmalogen 7.6%, sphingomyelin 10.8%, phosphatidylserine 7.1%, lysophosphatidylcholine 7.5%, phosphatidylinositol 3.1%, lysophosphatidylethanolamine 3.6%, phosphatidylinositol 4,5-bisphosphate 1.8%, phosphatidic acid 1.9%, phosphatidylinositol 4-phosphate 1.5%, and cardiolipin 1.9%. The cells possess high choline plasmalogen and lysophosphatidylethanolamine contents. The other phospholipids are within the normal biological ranges expected. Phospholipids were separated by high-performance liquid chromatography and quantified by lipid phosphorus assay.     

Phospholipid acyl group composition in normal and tumoral nerve cells in culture

We have studied the fatty acid composition of total phosphoglycerides from various types of nerve cells in culture. Primary cell cultures were compared with tumoral cell strains. Glial cells exhibited no characteristic pattern when compared to neurons. Tumoral cell phosphogly cerides contained much higher levels of octadecenoic acid and lower levels of C-20 to C-22 polyunsaturated fatty acids than normal cell phosphoglycerides. This observation seems to be a general feature in tumoral cell membranes. It could be of interest in respect to the membrane fluidity of cancer cells.     

Phospholipid oxidation in emulsions

The autoxidation of purified phosphatidyl ethanolamine (PE) and phosphatidyl choline (PC), extracted from egg and soybean lipids, was followed by oxygen uptake measurements in emulsified systems. All emulsified phospholipid fractions had comparable activation energies. Measurement by various physico-chemical tests was made of specific changes in the phospholipid molecule during autoxidation. PE oxidized more rapidly and absorbed more oxygen than PC. Higher 2-thiobarbituric acid test and diene and triene conjugation absorbance values were observed for PE than for PC. Of the two major polyunsaturated fatty acids in egg phospholipids, arachidonic acid disappeared at a more rapid rate during oxidation while the concentration of linoleic acid decreased to a level that was relatively constant. Although typical unsaturated fatty acid oxidation appeared to occur in all phospholipid fractions, oxidation in aqueous emulsions was only partly a function of fatty acid composition. The nitrogen moieties, ethanolamine and choline influenced the induction period for the oxidation of PE and PC respectively. Michigan Agriculture Experiment Station Journal Article No. 4420. Presented in part at the AOCS Meeting, Chicago, October 1967.     

Phospholipid composition ofCulex quinquefasciatus andCulex tritaeniorhynchus cells in logarithmic and stationary growth phases

Culex quinquefasciatus andCulex tritaeniorhynchus cells were grown in spinner culture and harvested in logarithmic and stationary phases of growth. The phospholipids were extracted from the cells, and the fatty acid profiles of the phospholipid classes were determined and compared. The major components were phosphatidylcholine and phosphatidylethanolamine, constituting≥80% of the phospholipid. The fatty acid profiles of lysophosphatidylcholine, phosphatidylinositol, and cardiolipin showed changes with aging of the Culex cells and between the species. In the lysophosphatidylcholine fraction, there was an increase in saturation of the fatty acids of theC. quinquefasciatus cells, and chain lengthening occurred in both species from the logarithmic to stationary phases of growth. In the phosphatidylinositiol fraction, both Culex species showed a decrease in monoenes and an increase in polyenes, while only theC. tritaeniorhynchus cells showed an increase in fatty acid chain length with aging. TheC. quinquefasciatus cells had an increase in polyenes with aging in the cardiolipin fraction. Differences in the percentage composition of the fatty acids were shown in all the phospholipid fractions between the Culex species in the logarithmic phase of growth and all except the phosphatidylinositiol and cardiolipin fractions in the stationary phase.     

纯磷脂单分子膜压缩过程中磷脂分子的构象和取向(英文)

On the basis of energy conservation law and surface pressure isotherm, the conformation energy changes of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) in pure phospholipid monolayer at the air/water interface during compression are derived. The optimized conformations of phospholipids at absolute freedom state are simulated by Gaussian 98 software. Based on following assumptions: (1)the conformation energy change is mainly caused by the rotation of one special bond; (2)the atoms of glycerol near the water surface are active; (3)the rotation is motivated by hydrogen-bond action; (4)the rotation of bond is inertial, one simplified track of conformational change is suggested and the conformations of DPPC and DPPG at different states are determined by the plots of conformation energy change vs. dihedral angle. The thickness of the simulated phospholipid monolayer is consistent with published experimental result. According to molecular areas at different states, the molecular orientations in the compressing process are also developed.     

厌氧水解的新解读：快速水解和慢速水解

挥发性脂肪酸（VFA）具有可生化性好、附加价值高等优点而得到广泛应用,厌氧消化产VFA是目前学术界的研究热点,但对有机物经过水解后的生物降解性能则少有论及,厌氧发酵过程中的水解产物存在“数量”和“质量”之间的权衡问题。为了进一步提高厌氧发酵过程中微生物对有机物的利用效率,突破水解对厌氧消化的限制性作用,从基质碳源释放快慢及厌氧发酵过程中碳源降解性能不同,本文将厌氧水解分为快速水解和慢速水解,分别阐述了两种水解方式的含义、分类以及优缺点,指出细胞内外碳源的释放速率和释放方式的不同是影响厌氧产酸和生物降解性能的决定性因素。最后指出快速水解与慢速水解相结合的分阶段联合处理方式,是今后厌氧消化产VFA的主要研究方向。     

Human placental lipid metabolism. III. Synthesis and hydrolysis of phospholipids

Both diacyl GPC (glycerylphosphorylcholine) and diacyl GPE (glycerylphosphorylethanolamine) are synthesized in human placental tissue from their respective monoacyl precursors. The origin of the monacyl phosphatides is apparently not the result of placental phosphatide acyl-hydrolase activity. The most likely source is maternal serum. The declining level of 1-acyl GPC in maternal serum is not attributable to lysophosphatide acylhydrolase activity and is probably explained by placental utilization for the synthesis of diacyl GPC.     

Control of lipid metabolism in cultured cells

Several studies are presented which indicate that composition of cell lipid is regulated by interaction between intracellular metabolism and lipid transport processes. When the fatty acid composition of cells cultured in essential fatty acid deficient conditions was studied, activation of synthesis of unusual polyun-saturated fatty acids was observed for a number of cell lines. In addition cells contained persistent residual amounts of linoleic acid, presumably owing to efficient scavenging mechanisms. The source of cell lipids was studied in both chemically defined and serum-supplemented media. In the absence of exogenous lipid, cells synthesize lipids from simple precursors, a process which is inhibited by adding serum. When serum lipid is present, cells preferentially utilize fatty acids as a source of nonsterol lipid. These are subsequently esterified intracellularly to make glycerides and phospholipids. When triglyceride is utilized as a source of cell lipid, it is first hydrolyzed before being taken up. By use of a nonhydrolyzable cholesterol ester analog, it is confirmed that both free and ester cholesterol are taken up and excreted by cells. Intracellular cholesterol content is thus regulated by rates of uptake, hydrolysis and excretion as well as by biosynthesis. One of 13 papers presented at the symposium “Lipid Metabolism in Cells in Culture,” AOCS Meeting, Houston, May 1971.     

Phospholipid exchange proteins in rat intestine

The 105,000 g supernatant and pH 5.1 supernatant fractions from rat intestinal homogenates stimulate phosphatidylcholine exchange between [³²P] phosphatidylcholine liposomes and beef heart mitochondria. This active fraction shows the characteristics of a protein. Isoelectric focusing of the intestinal pH 5.1 fraction shows two peaks of phosphatidylcholine exchange activity: one at an acidic pH (4.5–5.3), the other in a basic pH range (8–9). The second peak of activity appears to be a new phospholipid exchange protein. The anatomic distribution of phosphatidylcholine exchange activity in intestine has been investigated. Expressed per mg of protein, phosphatidylcholine exchange activity is higher in mucosa than in the intestinal wall. No significant differences have been found between villi and crypts cells or between jejunal and ileal villi. Furthermore, exchange activity per mg of protein in mucosa is unaffected by fasting or by feeding a high fat or high cholesterol diet. This suggests that phospholipid exchange activity in the absorptive cells is not a rate limiting step in the process of fat absorption. North Atlantic Treaty Organization grant recipient.     

Parathyroid hormone stimulates phosphatidylethanolamine hydrolysis by phospholipase D in osteoblastic cells

Parathyroid hormone (PTH) and phorbol-12,13-dibutyrate (PDBu) stimulate phospholipase D (PLD) activity and PC hydrolysis in UMR-106 osteoblastic cells {Singh, A.T., Kunnel, J.G., Strieleman, P.J., and Stern, P.H. (1999) Parathyroid Hormone (PTH)-(1–34), [Nle^{8, 18}, Tyr³⁴]PTH-(3–34) Amide, PTH-(1–31) Amide, and PTH-Related Peptide-(1–34) Stimulate Phosphatidylcholine Hydrolysis in UMR-106 Osteoblastic Cells: Comparison with Effects of Phorbol 12,13-Dibutyrate, Endocrinology 140, 131–137}. The current studies were designed to determine whether ethanolamine-containing phospholipids, and specifically PE, could also be substrates. In cells labeled with ¹⁴C-ethanolamine, PTH and PDBu treatment decreased ¹⁴C-PE. In cells co-labeled with ³H-choline and ¹⁴C-ethanolamine, PTH and PDBu treatment increased both ³H-choline and ¹⁴C-ethanolamine release from the cells. Choline and ethanolamine phospholipid hydrolysis was increased within 5 min, and responses were sustained for at least 60 min. Maximal effects were obtained with 10 nM PTH and 50 nM PDBu. Dominant negative PLD1 and PLD2 constructs inhibited the effects of PTH on the phospholipid hydrolysis. The results suggest that both PC and PE are substrates for phospholipase D in UMR-106 osteoblastic cells and could therefore be sources of phospholipid hydrolysis products for downstream signaling in osteoblasts.     

Phospholipid biosynthetic enzymes in human brain

Growing evidence suggests an involvement of brain membrane phospholipid metabolism in a variety of neurodegenerative and psychiatric conditions. This has prompted the use of drugs (e.g., CDPcholine) aimed at elevating the rate of neural membrane synthesis. However, no information is available regarding the human brain enzymes of phospholipid synthesis which these drugs affect. Thus, the objective of our study was to characterize the enzymes involved, in particular, whether differences existed in the relative affinity of substrates for the enzymes of phosphatidylethanolamine (PE) compared to those of phosphatidylcholine (PC) synthesis. The concentration of choline in rapidly frozen human brain biopsies ranged from 32–186 nmol/g tissue, a concentration similar to that determined previously for ethanolamine. Since human brain ethanolamine kinase possessed a much lower affinity for ethanolamine (K _m=460 μM) than choline kinase did for choline (K _m=17 μM), the activity of ethanolamine kinase in vivo may be more dependent on substrate availability than that of choline kinase. In addition, whereas ethanolamine kinase was inhibited by choline, and to a lesser extent by phosphocholine, choline kinase activity was unaffected by the presence of ethanolamine, or phosphoethanolamine, and only weakly inhibited by phosphocholine. Phosphoethanolamine cytidylyl-transferase (PECT) and phosphocholine cytidylyltransferase (PCCT) also displayed dissimilar characteristics, with PECT and PCCT being located predominantly in the cytosolic and particulate fractions, respectively. Both PECT and PCCT exhibited a low affinity for CTP (K_m approximately 1.2 mM), suggesting that the activities of these enzymes, and by implication, the rate of phospholipid synthesis, are highly dependent upon the cellular concentration of CTP. In conclusion, our data indicate different regulatory properties of PE and PC synthesis in human brain, and suggest that the rate of PE synthesis may be more dependent upon substrate (ethanolamine) availability than that of PC synthesis.     

Phospholipid liposomes: Preparation,characterization, and uses

Rhodopsin and cyclic guanosine monophosphat (cGMP)-dependent channel proteins are isolated from the rod outer segment disk membranes of dark-adopted bovine retinae and incorporated in liposomes, prepared by the method of detergent removal dialysis. The ion channel does not lose its transport function (release of Ca²⁺ ions by injection of cGMP) when incorporated in a liposome. Its activity depends on the degree of protein solubilization and the kind of detergent used. The highest activity is obtained by use of the detergent CHAPS. Shape, size, and size distribution of the liposomes are deduced from elastic and quasi-elastic light scattering, the liposome number density by viscometry, and the photopigment or Ca²⁺ content by optical absorbance. The liposomes are heterogeneous with respect to size and shape. Small unilamellar liposomes (R_h = 80 nm) and a narrow size distribution (U_D = 0.16) are obtained by using the detergent CHAPS. With increasing rhodopsin content per liposome, the hydrodynamic radius R_h increases and at the same time the shape of a liposome converts from a sphere to a prolate ellipsoid. The amount of entrapped Ca²⁺ per liposome reaches its maximum value when the Rhodopsin nearest-neighbor distance approaches its minimum value. This suggests an intermembrane protein-lipid-protein lattice, which serves as barriere for Ca²⁺. The influence of temperature or total used Ca²⁺ content is less profound. Increasing temperature yields slightly smaller liposomes.     

Increased Placental Phospholipid Levels in Pre-Eclamptic Pregnancies

Physiological pregnancy is associated with an increase in lipids from the first to the third trimester. This is a highly regulated response to satisfy energy and membrane demands of the developing fetus. Pregnancy disorders, such as pre-eclampsia, are associated with a dysregulation of lipid metabolism manifesting in increased maternal plasma lipid levels. In fetal placental tissue, only scarce information on the lipid profile is available, and data for gestational diseases are lacking. In the present study, we investigated the placental lipid content in control versus pre-eclamptic samples, with the focus on tissue phospholipid levels and composition. We found an increase in total phospholipid content as well as changes in individual phospholipid classes in pre-eclamptic placental tissues compared to controls. These alterations could be a source of placental pathological changes in pre-eclampsia, such as lipid peroxide insult or dysregulation of lipid transport across the syncytiotrophoblast.     

The hydrolysis of phosphatidylcholine by an immobilized lipase: Optimization of hydrolysis in organic solvents

The ability of a commercial immobilized lipase preparation (Lipozyme) to hydrolyze the fatty acyl ester bonds of soybean phosphatidylcholine in organic media was investigated. Response surface methodology, based on a Modified Central Composite design, was employed to examine the effects on hydrolysis of solvent polarity, water, pH, duration and temperature of incubation, and the amounts of substrate and catalyst. A second-order regression model was developed, which allows evaluation of the effects of these parameters, alone or in combination. Hydrolysis increased in a relatively straightforward manner in response to increases in incubation time and the amount of catalyst and was approximately constant over the range of substrate amounts studied. Solvent polarity had a profound effect on the degree of hydrolysis, and the qualitative and quantitative degrees of this effect were dependent upon the values of the other parameters studied. Conditions were identified where enzyme activity was strong in either nonpolar or polar solvents, with activity increasing as the polarity of the medium increased. Enzyme activity was minimum at about 37°C, increasing below and above this temperature. Activity was not affected by the presence of acid or base in the reactions. Increasing amounts of water stimulated enzyme activity in solvents more polar than hexane, while in less polar solvents water inhibited activity.     

Mitochondrial Glycerol-3-Phosphate Acyltransferase-Dependent Phospholipid Synthesis Modulates Phospholipid Mass and IL-2 Production in Jurkat T Cells

Changes in glycerophospholipid metabolism with age and disease can have a profound effect on immune cell activation and effector function. We previously demonstrated that glycerol‐3‐phosphate acyltransferase‐1, the first and rate limiting step in de novo glycerophospholipid synthesis, plays a role in modulating murine T cell function. The resultant phenotype is characterized by decreased IL‐2 production, increased propensity toward apoptosis, and altered membrane glycerophospholipid mass similar to that of an aged T cell. Since T cells in previous experiments were harvested from GPAT‐1^?/? mice, questions remained as to what extent the macro environment of the model influenced the observed cellular phenotype. Therefore, we generated and phenotypically characterized a mitochondrial glycerol‐3‐phosphate acyltransferase (GPAM) deficient Jurkat T cell. Furthermore, this line was used to probe possible mechanisms by which GPAT‐1/GPAM regulates T cell function. We report here that many of the key dysfunctional characteristics of murine GPAT‐1^?/? T cells are recapitulated in the GPAMKD Jurkat T cell. We found striking decreased IL‐2 production along with altered phospholipid mass and increased incidence of apoptosis. Since PtdOH is an indirect downstream product of GPAM, we attempted to rescue IL‐2 production with PtdOH supplementation; however, this addition did not return IL‐2 production to normal levels. Interestingly, we did find significantly decreased Zap‐70 phosphorylation following stimulation, suggesting that GPAM deficiency may alter membrane based stimulatory signaling. These data show for the first time that GPAM deficiency results in an inherent defect in Jurkat T cell function and glycerophospholipid composition and that this defect cannot be rescued by addition of exogenous PtdOH.     

Phospholipid Peroxidation: Lack of Effect of Fatty Acid Pairing

Phospholipids where both fatty acids are polyunsaturated are very rare. Most organisms prefer to couple their polyunsaturated fatty acids (PUFA) with either a saturated (SAT) or a monounsaturated (MUFA) fatty acid. This study examined if these natural couplings are there to protect PUFA from themselves. Specifically, does the coupling of PUFA to SAT or MUFA reduce the potential for increased rates of peroxidation by shrouding these highly peroxidisable fatty acids with less peroxidisable fatty acids? The influence of head group was examined by using the two most common phospholipids found in vertebrate membranes i.e. phosphatidylcholine and phosphatidylethanolamine species. Fatty acid pairings included 16:0/18:2 versus 18:2/18:2 and 16:0/22:6 versus 22:6/22:6. All phospholipids were incorporated into liposomes that were matched for their total PUFA content i.e. 25% PUFA/PUFA or 50% SAT/PUFA with phosphatidylcholine 16:0/16:0 used as the background phospholipid. An iron initiator (Fe²⁺/H₂O₂) was used to induce peroxidation and lipid hydroperoxide production was used to measure peroxidation. The results show that coupling of PUFA together on the same molecule does not increase peroxidation rates and therefore does not support the proposed hypothesis. The lower than expected levels of peroxidation measured for some phospholipid species (e.g. PtdEtn 22:6/22:6) is possibly due to the partitioning of these molecular species into the inner leaflet of the bilayer.     

Eicosapentaenoic and arachidonic acid: Comparison of metabolism and activity in murine epidermal cells

The biological activity, including metabolism and modulation of ornithine decarboxylase activity and DNA synthesis, of arachidonic acid (AA) and eicosapentaenoic acid (EPA) were compared in epidermal cells from SENCAR mice. Radiolabelled AA and EPA were found to be similarly incorporated into and released from membrane phospholipids of unstimulated cultures. However, when cells were stimulated with the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA), the release of AA was significantly higher than the release of EPA. The extent of metabolism of AA and EPA to prostaglandins was determined in both freeze-thawed cell preparations and in viable cultured cells. In the freeze-thawed preparations, use of AA as a substrate resulted in significantly more PGF than when EPA was used as the substrate. However, more PGE₃ was formed than PGE₂. PGD levels were the same for either fatty acid precursor. Prostaglandin production was also determined in viable cultured cells since other influences such as phospholipase A₂ activity can modify prostaglandin production. Control cultures prelabelled with either AA or EPA produced similar amounts of the respective PGF, PGE, and PGD. However, TPA-stimulated cultures produced significantly higher amounts of each prostaglandin in cultures prelabelled with AA compared to cells prelabelled with EPA. HETE or HEPE production was the same both for cultured cells prelabelled with AA or EPA and for homogenates from uncultured cells incubated directly with the radiolabelled fatty acids. TPA-induced ornithine decarboxylase (ODC) was significantly higher in AA-treated cultures compared to EPA-treated cultures. AA supports DNA synthesis to a greater extent than EPA, either alone or in the presence of TPA. These findings suggest that AA and EPA do not have equivalent biological activity in mouse epidermal cells.