Formation and function of phosphatidylserine and phosphatidylethanolamine in mammalian cells

Phosphatidylserine (PS) and phosphatidylethanolamine (PE) are metabolically related membrane aminophospholipids. In mammalian cells, PS is required for targeting and function of several intracellular signaling proteins. Moreover, PS is asymmetrically distributed in the plasma membrane. Although PS is highly enriched in the cytoplasmic leaflet of plasma membranes, PS exposure on the cell surface initiates blood clotting and removal of apoptotic cells. PS is synthesized in mammalian cells by two distinct PS synthases that exchange serine for choline or ethanolamine in phosphatidylcholine (PC) or PE, respectively. Targeted disruption of each PS synthase individually in mice demonstrated that neither enzyme is required for viability whereas elimination of both synthases was embryonic lethal. Thus, mammalian cells require a threshold amount of PS. PE is synthesized in mammalian cells by four different pathways, the quantitatively most important of which are the CDP-ethanolamine pathway that produces PE in the ER, and PS decarboxylation that occurs in mitochondria. PS is made in ER membranes and is imported into mitochondria for decarboxylation to PE via a domain of the ER [mitochondria-associated membranes (MAM)] that transiently associates with mitochondria. Elimination of PS decarboxylase in mice caused mitochondrial defects and embryonic lethality. Global elimination of the CDP-ethanolamine pathway was also incompatible with mouse survival. Thus, PE made by each of these pathways has independent and necessary functions. In mammals PE is a substrate for methylation to PC in the liver, a substrate for anandamide synthesis, and supplies ethanolamine for glycosylphosphatidylinositol anchors of cell-surface signaling proteins. Thus, PS and PE participate in many previously unanticipated facets of mammalian cell biology. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

Hybridoma perfusion systems: a comparison study

The efficiency of lgM production by hybridoma cells (1) cultured in suspension; (2) entrapped in alginate beads; or (3) packed in hollowfiber cartridge bioreactors, were compared in long-term perfusion cultures. The results showed that steady-state cell concentration and antibody production, per liter of perfused medium per day, were similar when cells were either entrapped in alginate beads of maintained in suspension. These values were also similar whether cells were maintained at high density in a hollowfiber cartridge bioreactro, or at low density in suspension. This work points out that cell behavior and antibody yield are comparable overall in the various perfusion systems currently used. However, a significant reduction of antibody production appeared whenever a part of the viable cells was lost in the filtrate. The reduction was due both to a decrease of viable cell yield and a decline of lgM productivity on a percell basis. This result is well in agreement with the previously presented model of "grow or die" cell cycle system of hybridoma, which proposes that the ratio of arrested to proliferating cells in perfusion cultures, should be increased in proportion to cell retention in the bioreactor, with a concomitant increase of lgM productivity.     

Disappearence of the intracytoplasmic membranes inRhodopseudomonas sphaeroides

The photosynthetic bacterium,Rhodopseudomonas sphaeroides, can be grown phototrophically (light, anaerobiosis), of chemotrophically (dark, aerobiosis). In the first case, it contains intracytoplasmic membranes with photosynthetic pigments. When shifted from phototrophy to chemotrophy these membranes disappear in an unknown fashion. In the present experiment, samples were taken for electron microscopy, cell density and bacteriochlorophyll determinations after shift from phototrophy to chemotrophy. The density of intracytoplasmic vesicles was measured on micrographs. During the first 2h growth is very slow and the ultrastructure remains unaltered. As growth resumes, the vesicles disappear at a rate which implies that they are not incorportated into the cytoplasmic membrane, nor actively digested, but remain intact and become increasingly diluted in the cytoplasm as the culture grows. The size of the vesicles was estimated to about 500 Å. The number of vesicles in phototrophically grown cells was calculated to about 575 per cell, and after 6h chemotrophic growth to about 100. The areas of the cytoplasmic and intracytoplasmic membranes are roughly calculated.Abbreviations Bchl bacteriochlorophyll - CM cytoplasmic membranes - ICM intracytoplasmic membranes     

Rapid flip-flop in polyunsaturated (docosahexaenoate) phospholipid membranes

The transbilayer movement (flip-flop) of 7-nitrobenz-2-oxa-1,3-diazol-4-yl phosphatidylethanolamine (NBD-PE) in phosphatidylcholine (PC) membranes containing various acyl chains was measured by dithionite quenching of NBD fluorescence. Of specific interest was docosahexaenoic acid (DHA), the longest and most unsaturated acyl chain commonly found in membranes. This molecule represents the extreme example of a family of important fatty acids known as omega-3s and has been clearly demonstrated to alter membrane structure and function. One important property that has yet to be reported is the effect of DHA on membrane phospholipid flip-flop. This study demonstrates that as the number of double bonds in the fatty acyl chains comprising the membrane increases, so does the rate of flip-flop of the NBD-PE probe. The increase is particularly marked in the presence of DHA. Half-lives t(1/2) of 0.29 and 0.086 h describe the process in 1-stearoyl-2-docosahexaenoylphosphatidylcholine and 1,2-didocosahexaenoylphosphatidylcholine, respectively, whereas in 1-stearoyl-2-oleoylphosphatidylcholine t(1/2)=11.5h. Enhanced permeability to dithionite with increasing unsaturation was also indicated by our results. We conclude that PC membranes containing DHA support faster flip-flop and permeability rates than those measured for other less-unsaturated PCs.     

Effect of lipid composition on rat liver nuclear membrane fluidity

Nuclear membrane fluidity is measured in rat liver by use of the fluorescence anisotropy of two probes: diphenylhexatriene and its cationic derivative trimethylammonium-diphenylhexatriene. It has been shown that, in 2-month-old rat liver cells, the bilayer surface is less fluid than the hydrophobic core. The fluidity was higher in 6-day-old rat liver nuclei, in which both the amount of cholesterol and the cholesterol/phospholipid ratio decreased. The influence of the single phospholipids, and in particular of phosphatidylcholine, has been studied by increasing the phosphatidylcholine with a choline base exchange reaction in isolated nuclear membranes. After this reaction, the fluorescence anisotropy of the bilayer surface increased, whereas at the hydrophobic core it decreased. Analysis of fatty acid composition shows an increase of phosphatidylcholine unsaturated fatty acids. The results show that the fluidity of nuclear membranes changes in relation to the lipid content and to the fatty acid composition. The role of nuclear membrane fluidity in cell function is discussed. © 1997 John Wiley & Sons, Ltd.     

Insect UV-, and green-photoreceptor membranes studied by the freeze-fracture technique

Summary The membranes of the microvilli of UV- and green-photoreceptors of the ant Myrmecia gulosa have been studied with the freeze-fracture technique. Both inner fracture faces, the cytoplasmic P-face and the extracellular E-face, are covered by globular particles. The P-face particles appear to be randomly distributed, occasionally forming clusters. Their density is about 7,000/m², and their mean diameter is 8.5 nm. The E-face particles, however, are arranged in an ordered square pattern with a center-to-center spacing of 9 nm. The density and distribution of P- and E-face particles are the same in both the UV- and the green-photoreceptor membranes. No differences were found in the ultrastructural organization of photoreceptor membranes after dark or light adaptation. It is suggested that the P-face particles represent rhodopsin molecules.     

Interaction of statins with phospholipid bilayers studied by solid-state NMR spectroscopy

Statins are drugs that specifically inhibit the enzyme HMG-CoA reductase and thereby reduce the concentration of low-density lipoprotein cholesterol, which represents a well-established risk factor for the development of atherosclerosis. The results of several clinical trials have shown that there are important intermolecular differences responsible for the broader pharmacologic actions of statins, even beyond HMG-CoA reductase inhibition. According to one hypothesis, the biological effects exerted by these compounds depend on their localization in the cellular membrane. The aim of the current work was to study the interactions of different statins with phospholipid membranes and to investigate their influence on the membrane structure and dynamics using various solid-state NMR techniques. Using ¹H NOESY MAS NMR, it was shown that atorvastatin, cerivastatin, fluvastatin, rosuvastatin, and some percentage of pravastatin intercalate the lipid-water interface of POPC membranes to different degrees. Based on cross-relaxation rates, the different average distribution of the individual statins in the bilayer was determined quantitatively. Investigation of the influence of the investigated statins on membrane structure revealed that lovastatin had the least effect on lipid packing and chain order, pravastatin significantly lowered lipid chain order, while the other statins slightly decreased lipid chain order parameters mostly in the middle segments of the phospholipid chains.     

Lysophosphoinositide-specific phospholipase C in rat brain synaptic plasma membranes

A membrane preparation from rat brain catalyzed the hydrolysis of [2-³H]glycerol-labeled lysophosphatidylinositol (lysoPI) to yield monoacylglycerol (MG) and inositolphosphates. This phospholipase C activity had an optimal pH of 8.2. The membrane preparation did not require the addition of Ca²⁺ for its maximum activity, but the activity was inhibited by addition of 0.1 mM EDTA to the assay mixture and was restored by simultaneous addition of 0.2 mM Ca²⁺. The activity was found to be localized in synaptic plasma membranes prepared by Ficoll and Percoll density gradients. The phospholipase C was highly specific for lysoPI; diacylglycerol formation from phosphatidylinositol, and MG formation from lysophosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidylserine were below 5% of that observed with lysoPI under the conditions used. We concluded that there is a pathway for phosphatidylinositol metabolism in brain synaptic membranes which is different from the well-characterized phosphoinositide-specific phospholipase C pathway.Abbreviations PI phosphatidylinositol - lysoPI lysophosphatidylinositol - lysoPI-PLC lysophosphoinositide-specific phospholipase C - PI-PLC phosphoinositide-specific phospholipase C - MG monoacylglycerol - PLC phospholipase C To whom to address reprint requests.     

SecA protein: Autoregulated initiator of secretory precursor protein translocation across theE. coli plasma membrane

Several classes ofsecA mutants have been isolated which reveal the essential role of this gene product forE. coli cell envelope protein secretion. SecA-dependent,in vitro protein translocation systems have been utilized to show that SecA is an essential, plasma membrane-associated, protein translocation factor, and that SecA's ATPase activity appears to play an essential but as yet undefined role in this process. Cell fractionation studies suggested that SecA protein is in a dynamic state within the cell, occurring in soluble, peripheral, and integral membraneous states. These data have been used to argue that SecA is likely to promote the initial insertion of secretory precursor proteins into the plasma membrane in a manner dependent on ATP hydrolysis. The protein secretion capability of the cell has been shown to translationally regulatesecA expression with SecA protein serving as an autogenous repressor, although the exact mechanism and purpose of this regulation need to be defined further.