Phospholipid Composition and Metabolism of Micrococcus denitrificans

The phospholipid composition of Micrococcus denitrificans was unusual in that phosphatidyl choline (PC) was a major phospholipid (30.9%). Other phospholipids were phosphatidyl glycerol (PG, 52.4%), phosphatidyl ethanolamine (PE, 5.8%), an unknown phospholipid (5.3%), cardiolipin (CL, 3.2%), phosphatidyl dimethylethanolamine (PDME, 0.9%), phosphatidyl monomethylethanolamine (PMME, 0.6%), phosphatidyl serine (PS, 0.5%), and phosphatidic acid (0.4%). Kinetics of ³²P incorporation suggested that PC was formed by the successive methylations of PE. Pulse-chase experiments with pulses of ³²P or acetate-1-¹⁴C to exponentially growing cells showed loss of isotopes from PMME, PDME, PS, and CL with biphasic kinetics suggesting the same type of multiple pools of these lipids as proposed in other bacteria. The major phospholipids, PC, PG, and PE, were metabolically stable under these conditions. The fatty acids isolated from the complex lipids were also unusual in being a simple mixture of seven fatty acids with oleic acid representing 86% of the total. Few free fatty acids and no non-extractable fatty acids associated with the cell wall or membrane were found.     

Phospholipid composition and metabolism of crustacean gills as related to changes in environmental salinities: relationship between Na+-K+-ATPase activity and phospholipids

The phospholipid composition and metabolism are studied in crustacean gills. It is reported that branchiae are rich in PC, PE and DPG and abundant in long chain polyunsaturated fatty acids (20:4 omega 6 and 20:5 omega 3 acids). The pathways of phospholipids synthesis appear similar to those described for vertebrates. It is demonstrated that there exist significant differences in the level of phosphatides between the anterior and posterior gills of Eriocheir sinensis. No matter what the salinity, the three more posterior located gills of Chinese crabs are shown to contain much more unsaturated phospholipids (PE and DPG). This is particularly true when animals are acclimated to dilute media. Moreover, lipids of posterior gills appear more fluid than the anterior ones as reported by the values of the degree of fluorescence polarization and the index of unsaturation of fatty acids. It is suggested that these lipid changes may indicate the existence of a functional difference between the various branchiae of euryhaline Eriocheir sinensis with respect to their ability to transport salt. It is shown that the renewal of DPG and PS is increased in posterior gills isolated from freshwater Chinese crabs. It is postulated that the enhanced formation of DPG in posterior gills is an indicator of an increased synthesis of mitochondria having as principal function to produce the necessary energy for the Na+ uptake. An attempt is made to correlate the PS metabolism and the Na+-K+-ATPase activity which is particularly located in the mitochondrial fractions of the three pairs of posterior gills.     

Effects of orally administered cytidine 5'-diphosphate choline on brain phospholipid content

Cytidine, as cytidine 5'-diphosphate choline (CDP-choline), is important for the synthesis of phosphatidylcholine in cell membranes. To investigate whether exogenous CDP-choline could affect brain phospholipid composition, we supplemented the diet of mice with this drug (500 mg/kg/day) for 27 months in 3-month-old mice and for 90, 42, and 3 days in 12-month-old mice, and measured their levels of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and the content of phosphatidylinositol plus phosphatidic acid in the cerebral cortex. After 27 months of treatment, PC and PE increased significantly by 19% (P < 0.05) and by 20% (P < 0.01), respectively. PS levels increased by 18% (not statistically significant). Similar elevations in PC and PE levels were obtained when older mice were treated for only 3 months (P < 0.05). No changes were observed with shorter treatment periods. These results suggest that chronic administration of CDP-choline can have effects on brain phospholipid composition that may underlie its reported utility in various neurologic disorders.     

Distribution of IP25 in chromatin and its possible involvement in chromatin condensation

Friend leukaemic cells (FLC) were induced to differentiate with dimethyl sulfoxyde (DMSO), hexamethylenbis-acetamide (HMBA) and sodium butyrate (SB) and the phospholipid composition was analyzed. The phospholipid composition of differentiated cells differed from that of non differentiated cells and also varied according to inducer. The ratios of the percentage of phosphatidyl choline (PC) to that of phosphatidyl ethanolamine (PE) or sphingomyelin (SPH) increased by about 2-fold in DMSO or SB induced FLC. These ratios did not vary in HMBa induced FLC. Furthermore the fatty acid composition of PC and PE obtained from differentiated cells varied according to the inducer. Although these changes appeared to be related to the inducers, it can not be excluded that the differentiated state also contributes to these changes.     

Ageing-Induced Alterations in Lipid/Phospholipid Profiles of Rat Brain and Liver Mitochondria: Implications for Mitochondrial Energy-Linked Functions

Effects of ageing on the lipid/phospholipid profile of brain and liver mitochondria from rats were examined. In the brain mitochondria the contents of total phospholipid (TPL) and cholesterol (CHL) increased with simultaneous increase in the TPL/CHL (mole:mole) ratio. The proportion and contents of lysophospholipid (Lyso), sphingomyelin (SPM), phosphatidylinositol (PI), phosphatidylserine (PS) and diphosphatidylglycerol (DPG) components increased, with maximal increases seen for PS and PI; phosphatidylcholine (PC) and phosphatidylethanolamine (PE) components registered decrease. In the liver mitochondria contents of TPL and CHL increased. However, the TPL/CHL (mole:mole) ratio was not altered. Lyso, PI and PS increased. However, the magnitude of increase was competitively lower; PE and DPG decreased. SPM and PC did not change as a consequence of ageing. These changes altered the contents of individual phospholipids in the two membrane systems. Respiration with glutamate, pyruvate + malate, succinate and ascorbate + N,N,N’,N’-tetramethyl-p-phenylenediamine was significantly impaired in brain mitochondria from old animals. For liver mitochondria the respiratory activity declined with glutamate and succinate. Correlation studies by regression analysis revealed that the lipid/phospholipid classes regulate respiratory function differently in the mitochondria from the two tissues. The respiration-related parameters in the brain mitochondria were dependent on multiple lipid/phospholipid components, and the process of regulation was complex compared to the liver mitochondrial functions.     

Compartmentation of phosphorylated precursors of phospholipid biosynthesis in cultured neuroblastoma cells

The continuous turnover of membrane phospholipids requires a steady supply of biosynthetic precursors. We evaluated the effects of decreasing extracellular Na+ concentration on phospholipid metabolism in cultured neuroblastoma (N1E 115) cells. Incubating cultures with 145 to 0 mM NaCl caused a concentration-dependent inhibition of [32P]phosphate uptake into the water-soluble intracellular pool and incorporation into phospholipid. Phospholipid classes were differentially affected; [32P]phosphate incorporated into phosphati-dylethanolamine (PE) and phosphatidylcholine (PC) was consistently less than into phosphatidylinositol (PI) and phosphatidylserine (PS). This could not be attributed to decreased phospholipid synthesis since under identical conditions, there was no effect on arachidonic acid or ethanolamine incorporation, and choline utilization for PC synthesis was increased. The effect of Na+ was highly specific since reducing phosphate uptake to a similar extent by incubating cultures in a phosphate-deficient medium containing Na+ did not alter the relative distribution of [32P]phosphate in phospholipid. Of several cations tested only Li+ could partially (50%) replace Na+. Incubation in the presence of ouabain or amiloride had no effect on [32P]phosphate incorporation into phospholipid. The differential effects of low Na+ on [32P]phosphate incorporation into PI relative to PC and PE suggests preferential compartmentation of [32P]phosphate into ATP in pools used for phosphatidic acid synthesis and relatively less in ATP pools used for synthesis of phosphocholine and phosphoethanolamine, precursors of PC and PE, respectively. This suggestion of heterogeneous and distinct pools of ATP for phospholipid biosynthesis, and of potential modulation by Na+ ion, has important implications for understanding intracellular regulation of metabolism.     

Incorporation of radioiodinated fatty acids into cardiac phospholipids of normoxic canine myocardium

The aim of this study was to assess the phospholipid distribution of radioiodinated 17-iodoheptadecanoic acid (IHDA), 15-(p-iodophenyl)pentadecanoic acid (p-IPPA) and 15-(p-iodophenyl)-3,3-dimethylpentadecanoic acid (DMIPPA) under normoxic conditions and to compare these data with the fatty acid composition of the phospholipid classes. After simultaneous i.v. injection of the radioiodinated fatty acids (1-123-IHDA; 1-131-p-IPPA; 1-125 DMIPPA) in open-chest dogs seven myocardial biopsies were taken over 40 min (n = 26). After lipid extraction of the biopsies the organic phase was analyzed for both neutral and polar lipids by two different TLC systems. The following polar lipid fractions were analyzed: lysophopshatidylcholine (LPC), sphingomyelin (SPH), phosphatidy1choline (PC; lecithin), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG; cardiolipin) and neutral lipids. Fractions were counted in a gamma well counter and corrected for cross-over and recovery. Results of the polar phospholipids analysis showed that IHDA has the highest incorporation into the phospholipids. The IHDA was mainly incorporated into PI (45.6%) followed by PC (30.9%), PE (14.0%) and PS (5.6%). The p-IPPA was predominantly incorporated incorporated into PC (37.2%), followed by PS (20.1%) and PE (13.7%). In contrast to IHDA, incorporation of p-IPPA into PI was small (6.4%). The DMIPPA analogue was incorporated into phopsholipids to only a very small degree, compared to IHDA and p-IPPA. PS (27.4%) was the only considerable phospholipid fraction into which DMIPPA was incorporated.The results clearly demonstrated that these radioiodinated fatty acid analogues have entirely different patterns of phospholipid incorporation. Major resemblances have been found between the incorporation into phospholipids of IHDA and the phospholipid distribution of the natural counterpart: stearic acid. The p—IPPA phospholipid incorporation only partly resembles the phospholipid distribution of palmitic acid. DMIPPA is because of its modified structure, incorporated into phospholipids to a low extent, mainly into PS. (Mol Cell Biochem116: 79–87, 1992)     

Composition and metabolism of phospholipids of human leukocytes

Human mononuclear (MN) and polymorphonuclear (PMN) leukocytes were analyzed for their phospholipid, triglyceride, cholesterol and fatty acid content. The phospholipid/cholesterol ratio was 1.24 for both cels. MN cells contain more phosphatidylcholine (PC), but less phosphatidylserine (PS), phosphatidylethanolamine (PE) and sphingomyelin (SPH) than PMN cells when expressed as percent of total phospholipid. When expressed on the basis of lipid content per cell, MN cells contain less PS, PE and SPH but more triglyceride than PMN cells. PMN cells incorporate palmitic, stearic, linoleic and linolenic acids into their phospholipids, triglycerides or cholesterol esters. The incorporation into triglycerides was highest for all fatty acids. Of the phospholipids, the incorporation was highest into PC. Labeled fatty acids also were found in proteins which had been delipidized by exhaustive extraction with organic solvents. These represent tightly or covalently bound fatty acids. The incorporation of [³H]palmitic acid into this protein fraction is stimulated by insulin.     

Effect of salinity on flavin-containing monooxygenase expression and activity in rainbow trout (Oncorhynchus mykiss)

In order to obtain more information about the physiological role(s) of flavin-containing monooxygenases (FMOs) in euryhaline teleost fishes, two experimental series were performed using adult and juvenile rainbow trout (Oncorhynchus mykiss). Cannulated adult trout were exposed to freshwater or 21% seawater for 48 h, whereas juvenile trout were acclimated to one of four different salinities: freshwater, 7%, 14%, or 21% during a 2-week period. FMO expression and activity were determined in red blood cells (RBC), liver, gill, kidney, gut, heart and brain. Furthermore, the content of trimethylamine oxide (TMAO; an FMO metabolite and an osmolyte) as well as urea were determined in various tissues. FMO expression and activity increased significantly and in a salinity dependent manner in osmoregulatory organs (gills, kidney and gut) in both juveniles and adult trout and, furthermore, in RBC in adults. No significant changes were observed in liver or heart. Urea content increased significantly and in a salinity dependent manner in all tissues, whereas TMAO was accumulated primarily in muscle tissue. Salinity dependent adjustment of FMO expression and activity primarily in osmoregulatory organs as well as regulation of TMAO content in muscle is consistent with previous studies showing an association of FMO with osmoregulation in euryhaline teleosts. However, the lack of a parallel increase of TMAO with urea in other tissues of fish at high salinity indicates other mechanisms of protection from intracellular urea may exist in non-muscular tissues.     

Changes in phospholipids and free fatty acids in the brains of mice preconditioned by hypoxia.

Changes in the composition and contents of phospholipids and free fatty acids were observed and compared in three groups: (A) unpreconditoned normal controls, (B) exposure to 1 run of hypoxia and (C) exposure to 4 runs of hypoxia. In group B, the content of phosphatidyl ethanolamine (PE), phosphatidyl serine (PS) and free fatty acids (FFAs) increased significantly and the content of phosphatidyl choline (PC) and sphingomyelin (SM) decreased significantly. While in group C the content of PE, PS, PC and FFAs changed significantly when compared with that of group B, all phospholipid (except SM) and FFA contents tended to decrease to the level of group A. No new FFA was seen in the brain homogenates in any of the three groups. These results suggest that the changes in the content of mouse brain phospholipids and FFAs may be adaptive and involved in the animals' tolerance to hypoxia.     

Effect of cryopreservation on lipids and some physiological features of spermatozoa from rams pastured in highlands and in valleys

The effect of low temperature preservation on the motility and morphology of acrosomes, acrosomal proteolytic activity, phospholipid and fatty acid composition of phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE), and the cholesterol/phospholipid molar ratio in sperm from rams housed in the highlands or in the valleys, were studied. The indices of motility and morphological integrity of sperm from highland rams were much greater compared with those of valley rams. Phosphatidyl choline (PC) of the highland rams was more unsaturated, while PE was more saturated compared with those of valley rams. Cryopreservation of the sperm from highland rams significantly increased the content of choline plasmalogen, accompanied by a slight rise in the levels of lysophosphatidyl choline (LPC) and phosphatidyl inositol (PI) in their sperm. The fatty acid composition altered following cryopreservation. These variations were mainly due to a decrease in the amount of docosahexaenic acid and an increase in the amounts of linoleic and palmitic fatty acids. The results may be indicative of the fact that the alterations in the sperm of the valley rams were more pronounced and they may be attributed to the structural features of the sperm, as well as a reduced concentration of oxygen in the organs and tissues of the highland rams.     

Importance of phosphatidylethanolamine for association of protein kinase C and other cytoplasmic proteins with membranes.

Biological membranes exhibit an asymmetric distribution of phospholipids. Phosphatidylserine (PS) is an acidic phospholipid that is found almost entirely on the interior of the cell where it is important for interaction with many cellular components. A less well understood phenomenon is the asymmetry of the neutral phospholipids, where phosphatidylcholine (PC) is located primarily on exterior membranes while phosphatidylethanolamine (PE) is located primarily on interior membranes. The effect of these neutral phospholipids on protein-phospholipid associations was examined using four cytoplasmic proteins that bind to membranes in a calcium-dependent manner. With membranes containing PS at a charge density characteristic of cytosolic membranes, protein kinase C and three other proteins with molecular masses of 64, 32, and 22 kDa all showed great selectively for membranes containing PE rather than PC as the neutral phospholipid; the calcium requirements for membrane-protein association of the 64- and 32-kDa proteins were about 10-fold lower with membranes containing PE; binding of the 22-kDa protein to membranes required the presence of PE and could not even be detected with membranes containing PC. Variation of the PS/PE ratio showed that membranes containing about 20% PS/60% PE provided optimum conditions for binding and were as effective as membranes composed of 100% PS. Thus, PE, as a phospholipid matrix, eliminated the need for membranes with high charge density and/or reduced the calcium concentrations needed for protein-membrane association. A surprising result was that PKC and the 64- and 32-kDa proteins were capable of binding to neutral membranes composed entirely of PE/PC or PC only. The different phospholipid headgroups altered only the calcium required for membrane-protein association. For example, calcium concentrations at the midpoint for association of the 64-kDa protein with membranes containing PS, PE/PC, or PC occurred at 6, 100, and 20,000 microM, respectively. Thus, biological probes detected major differences in the surface properties of membranes containing PE versus PC, despite the fact that both of these neutral phospholipids are often thought to provide "inert" matrices for the acidic phospholipids. The selectivity for membranes containing PE could be a general phenomenon that is applicable to many cytoplasmic proteins. The present study suggested that the strategic location of PE on the interior of the membranes may be necessary to allow some membrane-protein associations to occur at physiological levels of calcium and PS.     

31P NMR of tissue phospholipids: a comparison of three tissue pre-treatment procedures

Extracted tissue phospholipid 31P NMR profiles, obtained from individual porcine lenses subjected to two preservation procedures (acetone desiccation and freeze-drying) and a perchloric acid-extraction procedure, were compared to those from freshly excised lens specimens. Each profile yielded quantitative data on 12 lens phospholipids: PC, LPC, PC plas, PE, LPE, PE plas, PS, SPH, PI, LPI, PG, and CL. A specimen group size of at least 9 lenses was required for secure statistical inter-group comparisons by the Scheffé procedure, due to specimen 31P NMR profile variability, interpreted as arising from specimen biological variability. The phospholipid profiles of lenses preserved by acetone desiccation were essentially identical to those from the freshly excised control lenses. Freeze-dried lens profiles differed significantly in four components, while profiles from perchloric acid-extracted lenses differed in six. It is concluded that specimen preservation by acetone disiccation is a useful method for preserving tissue phospholipids for subsequent 31P NMR profile analysis, while freeze-drying is not. Lipid extraction following a tissue acid extraction is also of little or no value in the determination of tissue phospholipid profiles.     

Lipids from nerve tissues of the horseshoe crab, Limulus polyphemus.

1. The predominant lipids of nerve cords, ganglion and brain from horseshoe crabs were cholesterol (11% of lipid) and phospholipid (81% of lipid). 2. Major phospholipids were phosphatidyl ethanolamine and phosphatidyl choline with lesser amounts of phosphatidyl serine and phosphatidyl inositol and sphingomyelin. 3. The phospholipid fraction was characterized by a high content of plasmalogen, i.e. alk-1-enyl acyl phosphatides, so that 42% of the ethanolamine phosphatides were the plasmalogen, phosphatidal ethanolamine. 4. Phosphatidyl choline and phosphatidyl ethanolamine were high in polyunsaturation with 20:4 and 20:5 major fatty acids. Sphingomyelin had predominantly long chain saturated fatty acids. 5. Cerebrosides and gangliosides, which are associated with vertebrate nerve tissues, were absent from nerves of horseshoe crabs.     

The interaction of cytochrome C with phospholipids. A pulse-radiolysis study.

The reactions of the hydrated electron (eaq-), produced during pulse radiolysis, have been used to study the binding of phosphatidyl choline (PC), phosphatidyl serine (PS), phosphatidyl ethanolamine (PE), and phosphatidyl inositol (PI) vesicles with horse-heart cytochrome C. An interaction could only be detected between cytochrome C and either PS or PI. An apparent equivalence point in the binding was reached for both phospholipids at a molar ratio of phospholipid : protein of 6 : 1. At this point, the reactivity of (eaq-) towards the cytochrome C was very markedly reduced. Indeed, the rate of disappearance of (eaq-) under such conditions was the same as the rate of eaq- disappearance in triply-distilled water. The inclusion of cholesterol at a molar ratio of 1 : 1 within the phospholipid vesicles changed the stoichiometry of the interaction. Evidence that protonated epsilon-amino groups of lysine residues are involved in the interaction is presented. Possible models for the complexes formed are discussed.     

Changes in phospholipid composition of Thiobacillus neapolitanus during growth

Summary During the stationary growth phase, the phospholipids of Thiobacillus neapolitanus consisted of phosphatidyl glycerol (PG), diphosphatidyl glycerol (DPG), phosphatidyl-N-monomethylethanolamine (PME) and phosphatidyl ethanolamine (PE) in increasing amounts. In general, the phospholipids increased to a maximum concentration during the stationary phase and then decreased in concentration. Individually, PG and PE increased to a maximum in late lag or early exponential phase and then decreased in concentration. DPG and PME increased during the transition between the exponential and the stationary phase and reached a maximum concentration in the stationary phase. In older cultures, a quantitative interconversion between PG and DPG and PE and PME was observed. A lyso-phospholipid compound also appeared in the late stationary phase.The phospholipid composition of the culture supernatant fluid was essentially similar to that of the cells at all stages of growth. No excessive secretion of these products into the medium was observed at any growth stage of the culture.Abbreviations used PG Phosphatidyl glycerol - DPG Diphosphatidyl glycerol - PME Phosphatidyl-N-monomethylethanolamine - PE Phosphatidyl ethanolamine - GPGPG Glycerophosphoryl glycerophosphoryl glycerol - GPG Glycerophosphoryl glycerol - GPE Glycerophosphoryl ethanolamine - GPME Glycerophosphoryl-N-monomethylethanolamine     

Distribution of omega-3 and omega-6 fatty acids in the ether- and ester-linked phosphoglycerides from tissues of the rainbow trout, Salmo gairdneri

1. Data presented here demonstrate that polyunsaturated fatty acids in the phospholipids of rainbow trout tissues are compartmentalized differently than in mammalian tissues. 2. We have determined the distribution of omega-3 (n-3) and omega-6 (n-6) fatty acids in the alkyl-, alk-1-enyl-, and diacyl- subclasses of phosphatidylcholines (PC), phosphatidyl-ethanolamines (PE), phosphatidylinositols (PI), and phosphatidylserines (PS) from gill, kidney and spleen of rainbow trout. 3. Alkyl-linked PC and alk-1-enyl-linked PE were the most abundant ether-containing phospholipids, amounting to 10-15% of each class; no ether-linked PI or PS was detected. 4. C20:4 n-6 was found in high concentrations only in PI; the n-3 fatty acids were found in highest concentration in the ether-linked phospholipids as compared with the diacyl subclasses and C20:5 n-3 was especially prevalent in 1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine and C22:6 n-3 was prevalent in PS.     

Aquaporin molecular characterization in the sea-bass (Dicentrarchus labrax): the effect of salinity on AQP1 and AQP3 expression

Euryhaline fish possess the ability to compensate for environmental salinity changes through hydro-mineral regulation. A number of proteins have been studied in order to understand water and ion exchanges, known as fish osmoregulation. Sea-bass (Dicentrarchus labrax) cDNA sequences encoding a homologue of mammalian aquaporin (termed AQP1) and a homologue of mammalian aquaglyceroporin (termed AQP3) have been isolated and sequenced. The aquaporin amino acid sequences share respectively more than 60% and 65% identity with other known aquaporins. We have shown that salinity influences aquaporin expression levels in the gill, kidney and digestive tract, the main osmoregulatory organs. AQP1 may have a major osmoregulatory role in water transport in kidney and gut in SW-acclimated fish, whereas AQP3 could be implicated in gill water transport in FW-acclimated fish.     

The Effects of Acetylcholine on the Turnover of Phosphatidic Acid and Phosphoinositide in Sympathetic Ganglia, and in Various Parts of the Central Nervous System in Vitro

The effect of acetylcholine on the incorporation of P³² into the individual phosphatides in slices of various structures of the nervous system has been studied. There was a marked stimulation of P³² incorporation into phosphoinositide and phosphatidic acid, but not into phosphatidyl choline and phosphatidyl ethanolamine, in the cat stellate and celiac ganglia in vitro. Acetylcholine stimulated P³² incorporation into certain phosphatides, primarily phosphoinositide and phosphatidic acid, in several structures of the cat and guinea pig brain; there was little or no effect of acetylcholine on phosphatide turnover in the inferior corpora quadrigsemina and cerebellar cortex. The suggestion is made that the phospholipid effect can best be explained as being concerned with the active transport of sodium ions out of the cell across the postsynaptic membrane of cholinergic neurons in response to acetylcholine.