Phospholipid metabolism in ehrlich ascites tumor cells. II. Turnover rate of ether phospholipids.
(17/2589)
1. Radioactive precursors of phospholipids, i.e., 32Pi, [1-14C]glycerol, [2-3H]glycerol, and [1-14C]acetate, were individually injected into the peritoneal cavity of mice bearing Ehrlich ascites tumor cells and the rates of incorporation were estimated. 2. Although [2-3H]glycerol was not practically incorporated into ether phospholipids, the other three radioactive precursors were incorporated into diacyl, 1-O-alkenyl-2-acryl-, and 1-O-alkyl-2-acryl-GPE (GPC). 3. In the experiments on 32Pi or [1-14C]acetate incorporation, 1-O-alkyl compounds in the ethanolamine phosphoglyceride fraction showed high specific activities in comparison with 1-acyl compounds. In the case of [1-14C]glycerol incorporation, a high rate of incorporation into 1-O-alkyl compounds was not found. In the choline phosphoglyceride fraction, a high rate of incorporation of the above precursors into 1-O-alkyl compounds was not observed. 4. The specific activities of 1-O-alkenyl compounds were fairly low compared with those of 1-acyl- and 1-O-acyl- and 1-O-alkyl compounds throughout the incorporation experiments with [1-14C]glycerol and [1-14C]acetate, but in 32Pi incorporation, 1-O-alkenyl compounds showed higher specific activities than 1-acyl compounds in ethanolamine phosphoglyceride, suggesting an exchange reaction of the phosphorylethanolamine moiety. 5. From the above findings, it appears that alkyl ether phospholipids of ethanolamine from may have a significant role in ascites tumor cells, based on their rapid turnover. (+info)
Phospholipid composition of Rickettsia prowazeki grown in chicken embryo yolk sacs.
(18/2589)
The phospholipid composition and phospholipid fatty acid composition of purified Rickettsia prowazeki were determined. The lipid phosphorous content was 6.8 +/- 1.3 microgram/mg of total rickettsial protein. The major phospholipid was phosphatidylethanolamine (60 to 70%); phosphatidylglycerol constituted 20%, and phosphatidylcholine constituted 15%. Small amounts of phosphatidylserine and cardiolipin were detected. The principal fatty acids were 18:1, 16:1, and 16:0. The fatty acid composition of the phosphatidylcholine in the rickettsial extracts was very different than that of the other rickettsial phosphatides and very similar to that of normal yolk sac phosphatidylcholine. The specific of the phosphatidylcholine of rickettsiae grown in the presence of 32P was markedly lower than that of phosphatidylethanolamine and phosphatidylglycerol. It is suggested that the phosphatidylcholine in the rickettsial extract is yolk sac derived and either tightly absorbed or exchanged into the rickettsial membrane. (+info)
Total plasmalogens and O-(acylalkylglycerophosphoryl) ethanolamine from labelled hexadecanol and palmitate during hypoxia and anoxia in rat heart.
(19/2589)
By the use of the Langendorff technique, surviving isolated rat hearts were perfused with [1-14 C] palmitate, [1-14C] hexadecanol or [1-14C,1-3H] hexadecanol under normal or anoxic conditions. After perfusion for 30min with either precursor, when oxygenated or in an hypoxic condition, or when 1mM-KCN was included in the system, the heart tissues showed no significant chemical changes in their content of total lipids, total phospholipids or total ethanolamine-containing phospholipids. Changes were observed in the ratio of alkyl-to alk-1-enyl-glycerophosphorylethanolamine in the tissue perfused with N2+CO1 plus CN-. A slight increase from 4.0+/-0.3 to 4.9+/-0.2% in alkyl derivatives and a decrease from 11.2+/-0.4 to 9.4+/-0.3% in alk-1-enyl derivatives was observed. The incorporation of the [14C] palmitate and the [14C] hexadecanol into the recovered phospholipids and plasmalogens was severely decreased in the tissues perfused with CN-: in the hypoxic state only a mild inhibition was observed compared with the oxygenated systems. Considerable 3H from [1-14C, 1-3H] hexadecanol was retained (25-35%) in the alk-1-enylether chains of plasmalogens under both the oxygenated conditions and with CN-, suggesting that the same mechanism of incorporation is operational at high or low O2 concentrations. The results are consistent with an O2-dependent, CN-sensitive step in the biosynthesis of plasmalogens in the rat heart. (+info)
Cloning and expression of a human choline/ethanolaminephosphotransferase: synthesis of phosphatidylcholine and phosphatidylethanolamine.
(20/2589)
Cholinephosphotransferase catalyses the final step in the synthesis of phosphatidylcholine (PtdCho) via the Kennedy pathway by the transfer of phosphocholine from CDP-choline to diacylglycerol. Ethanolaminephosphotransferase catalyses an analogous reaction with CDP-ethanolamine as the phosphobase donor for the synthesis of phosphatidylethanolamine (PtdEtn). Together these two enzyme activities determine both the site of synthesis and the fatty acyl composition of PtdCho and PtdEtn synthesized de novo. A human choline/ethanolaminephosphotransferase cDNA (hCEPT1) was cloned, expressed and characterized. Northern blot analysis revealed one hCEPT1 2.3 kb transcript that was ubiquitous and not enriched, with respect to actin, in any particular cell type. The open reading frame predicts a protein (hCEPT1p) of 416 amino acid residues with a molecular mass of 46550 Da containing seven membrane-spanning domains. A predicted amphipathic helix resides within the active site of the enzyme with the final two aspartic residues of the CDP-alcohol phosphotransferase motif, DG(X)2AR(X)8G(X)3D(X)3D, positioned within this helix. hCEPT1p was successfully expressed in a full-length, active form in Saccharomyces cerevisiae cells devoid of endogenous cholinephosphotransferase or ethanolaminephosphotransferase activities (HJ091, cpt1::LEU2 ept1-). In vitro, hCEPT1p displayed broad substrate specificity, utilizing both CDP-choline and CDP-ethanolamine as phosphobase donors to a broad range of diacylglycerols, resulting in the synthesis of both PtdCho and PtdEtn. In vivo, S. cerevisiae cells (HJ091, cpt1::LEU2 ept1-) expressing hCEPT1 efficiently incorporated both radiolabelled choline and ethanolamine into phospholipids, demonstrating that hCEPT1p has the ability to synthesize both choline- and ethanolamine- containing phospholipids in vitro and in vivo. (+info)
Transbilayer movement and distribution of spin-labelled phospholipids in the inner mitochondrial membrane.
(21/2589)
The transmembrane diffusion and equilibrium distribution of spin-labelled phosphatidylethanolamine (PE*), phosphatidylcholine (PC*) and cardiolipin (CL*) were investigated in purified mitochondrial inner membranes using electron spin resonance spectroscopy. Using the back exchange technique, we found that the outside-inside movement of PE* and PC* in beef-heart inner mitochondrial membranes was rapid (t1/2 in the range 10-15 min at 30 degrees C). The steady-state distributions in non-energised mitoplasts were approximately 30% in the inner leaflet for PC* and 39% for PE*. Within the limits of probe concentration that can possibly be used in these experiments, the initial velocity of the inward movement was not saturable with respect to the amount of analogue added to the membranes, suggesting that the spin-labelled phospholipids diffused passively between the two leaflets of the inner mitochondrial membrane. In energised mitoplasts, PC* behaviour was not affected, PE* diffused approximately two times faster toward the inner monolayer but reached the same plateau. Treatment of energised mitochondria with N-ethylmaleimide did not affect PC* diffusion, while the kinetics of PE* internalisation became identical to that of PC*. Similar results were found when PC* and PE* movements were studied in mitoplasts from beef heart, rat liver or yeast. The spin-labelled cardiolipin, which possesses four long chains, had to be introduced in the mitoplast with some ethanol. After equilibration (t1/2 of the order of 13 min at 30 degrees C), the transmembrane distribution suggested that approximately half of the cardiolipin analogue remained in the outer leaflet. These results do not allow us to determine if a specific protein (or flippase) is involved in the phospholipid transmembrane traffic within inner mitochondrial membranes, but they show that lipids can rapidly flip through the mitochondrial membrane. (+info)
Nisin promotes the formation of non-lamellar inverted phases in unsaturated phosphatidylethanolamines.
(22/2589)
Nisin, a peptide used as a food preservative, is shown, by 31P-nuclear magnetic resonance and infrared spectroscopy, to perturb the structure of membranes formed of unsaturated phosphatidylethanolamine (PE) and to induce the formation of inverted non-lamellar phases. In the case of dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), nisin promotes the formation of inverted hexagonal phase. Similarly, the peptide induces the formation of an isotropic phase, most likely a cubic phase, with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE). It is proposed that the insertion of the peptide in the bilayer shifts the amphiphilic balance by increasing the hydrophobic contribution and is at the origin of the changes in the polymorphic propensities of PE. This is supported by the fact that the presence of cholesterol in the PE bilayer inhibits the power of nisin to perturb the membrane structure, most likely because the peptide insertion is difficult in the fluid ordered phase. This finding provides insight into possible antibacterial mechanisms of nisin. (+info)
Docosahexaenoic acid (DHA) alters the phospholipid molecular species composition of membranous vesicles exfoliated from the surface of a murine leukemia cell line.
(23/2589)
Previously, we presented evidence that the vesicles routinely exfoliated from the surface of T27A tumor cells arise from vesicle-forming regions of the plasma membrane and possess a set of lateral microdomains distinct from those of the plasma membrane as a whole. We also showed that docosahexaenoic acid (DHA, or 22:6n-3), a fatty acyl chain known to alter microdomain structure in model membranes, also alters the structure and composition of exfoliated vesicles, implying a DHA-induced change in microdomain structure on the cell surface. In this report we show that enrichment of the cells with DHA reverses some of the characteristic differences in composition between the parent plasma membrane and shed microdomain vesicles, but does not alter their phospholipid class composition. In untreated cells, DHA-containing species were found to be a much greater proportion of the total phosphatidylethanolamine (PE) pool than the total phosphatidylcholine (PC) pool in both the plasma membrane and the shed vesicles. After DHA treatment, the proportion of DHA-containing species in the PE and PC pools of the plasma membrane were elevated, and unlike in untreated cells, their proportions were equal in the two pools. In the vesicles shed from DHA-loaded cells, the proportion of DHA-containing species of PE was the same as in the plasma membrane. However, the proportion of DHA-containing species of PC in the vesicles (0.089) was much lower than that found in the plasma membrane (0.194), and was relatively devoid of species with 16-carbon acyl components. These data suggested that DHA-containing species of PC, particularly those having a 16-carbon chain in the sn-1 position, were preferentially retained in the plasma membrane. The data can be interpreted as indicating that DHA induces a restructuring of lateral microdomains on the surface of living cells similar to that predicted by its behavior in model membranes. (+info)
The influence of vitamin K1 on the structure and phase behaviour of model membrane systems.
(24/2589)
Vitamin K1 is a component of the Photosystem I of plants which constitutes the major dietary form of vitamin K. The major function of this vitamin is to be cofactor of the microsomal gamma-glutamylcarboxylase. Recently, novel roles for this vitamin in the membrane have been postulated. To get insight into the influence of vitamin K1 on the phospholipid component of the membrane, we have studied the interaction between vitamin K1 and model membranes composed of dimyristoylphosphatidylcholine (DMPC) and dielaidoylphosphatidylethanolamine (DEPE). We utilized high-sensitivity differential scanning calorimetry and small-angle X-ray diffraction techniques. Vitamin K1 affected the thermotropic properties of the phospholipids, broadened and shifted the transitions to lower temperatures, and produced the appearance of several peaks in the thermograms. The presence of the vitamin gave rise to the formation of vitamin-rich domains which were immiscible with the bulk phospholipid in both the gel and the liquid-crystalline phases. Vitamin K1 was unable to alter the lamellar organization of DMPC, but we found that it produced an increase in the interlamellar repeat spacing of DMPC at 10 degrees C. Interestingly, vitamin K1 promoted the formation of inverted hexagonal HII structures in the DEPE system. We discuss the possible implications that these vitamin K1-phospholipid interactions might have with respect to the biological function of the vitamin. (+info)