Stimulation of phospholipase A2 activity by high osmotic pressure on cholesterol-containing phospholipid vesicles

Bovine serum albumin conjugates of guanosine prepared by the periodate method was used as immunogen to elicit guanosine antibodies in rabbits. The specificities of the antibodies were studied by the inhibition of their binding to [3H]Gox-red, [32P]DNA and [3H]RNA by related non-radioactive compounds. A population of antibodies is specific to Gox-red with an average association constant of around 10(7) M-1 at 4 degrees C. There are a population of antibodies which bind to [32P]ssDNA and [3H]RNA specifically at guanosine residues. RNA binding antibodies were separated into two populations by affinity chromatography.     

Membrane-associated cytoskeleton and transbilayer phospholipid asymmetry

Earlier studies have suggested that the membrane-associated cytoskeleton (membrane skeleton) in erythrocytes plays a major role in maintaining the transmembrane phospholipid asymmetry. But recently, it has been proposed that an ATP-dependent aminophospholipid pump is the sole determinant of this asymmetry in these cells. A critical analysis of the published data along with some unpublished results from the author's laboratory, however, indicate that both membrane skeleton and ATP-dependent aminophospholipid pump are required for maintaining the membrane phospholipid asymmetry in native erythrocytes.     

Lateral pressure dependence of the phospholipid transmembrane diffusion rate in planar-supported lipid bilayers

The dependence of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) flip-flop kinetics on the lateral membrane pressure in a phospholipid bilayer was investigated by sum-frequency vibrational spectroscopy. Planar-supported lipid bilayers were prepared on fused silica supports using the Langmuir-Blodgett/Langmuir-Schaeffer technique, which allows precise control over the lateral surface pressure and packing density of the membrane. The lipid bilayer deposition pressure was varied from 28 to 42 mN/m. The kinetics of lipid flip-flop in these membranes was measured by sum-frequency vibrational spectroscopy at 37°C. An order-of-magnitude difference in the rate constant for lipid translocation (10.9 × 10⁻⁴ s⁻¹ to 1.03 × 10⁻⁴ s⁻¹) was measured for membranes prepared at 28 mN/m and 42 mN/m, respectively. This change in rate results from only a 7.4% change in the packing density of the lipids in the bilayer. From the observed kinetics, the area of activation for native phospholipid flip-flop in a protein-free DPPC planar-supported lipid bilayer was determined to be 73 ± 12 Å²/molecule at 37°C. Significance of the observed activation area and potential future applications of the technique to the study of phospholipid flip-flop are discussed.     

Calcium, phospholipid turnover and transmembrane signalling

Turnover of phosphatidylinositol, which is provoked by various neurotransmitters, peptide hormones and many other biologically active substances, appears to serve as a signal for the transmembrane control of protein phosphorylation through activation of a novel protein kinase (C-kinase). The activation of this enzyme absolutely requires Ca2+ and phosphatidylserine. Diacylglycerol derived from the receptor-linked breakdown of phosphatidylinositol dramatically increases the affinity of C-kinase for Ca2+, and thereby renders this enzyme fully active without a net increase in the concentration of Ca2+. Under appropriate conditions synthetic diacylglycerol directly added to intact cell systems activates C-kinase fully without interaction with surface receptors. By using such synthetic diacylglycerol and the Ca2+ ionophore A23187, it is shown that either receptor-linked protein phosphorylation or Ca2+ mobilization alone is merely a prerequisite but not a sufficient requirement, and both are synergistically effective for causing a full physiological cellular response. In some tissues cyclic nucleotides, both cyclic AMP and cyclic GMP, may inhibit the receptor-linked breakdown of phosphatidylinositol, and appear to provide negative control that prevents over-response.     

Endocytosis of glycophospholipid-anchored and transmembrane forms of CD4 by different endocytic pathways.

A glycophosphatidylinositol (GPI)-anchored form of the CD4 receptor was constructed by fusing the extracellular domain of CD4 to the COOH-terminus of decay accelerating factor (DAF), containing a signal for GPI-anchor attachment. The internalization of GPI-linked CD4 (CD4DAF) was compared to that of transmembrane CD4 using both [125I]gp120 and anti-CD4 antibodies. We show that transmembrane CD4 is rapidly endocytosed in transfected CHO cells, while CD4DAF is internalized at a rate approximately 3-fold slower. Immunoelectron microscopy suggests that whereas transmembrane CD4 is endocytosed via clathrin-coated vesicles, CD4DAF enters cells by an alternative pathway involving non-coated microinvaginations of the plasma membrane. Following internalization CD4DAF recycles through a primaquine-insensitive compartment, whereas the recycling of transmembrane CD4 is inhibited by primaquine, suggesting that the two receptors may recycle from distinct populations of early endosomes. Colocalization of both CD4DAF and CD4 with an antibody against a lysosomal membrane protein suggests that the two endocytic pathways may converge.     

Enzyme and phospholipid asymmetry in liver microsomal membranes

The transverse distribution of enzyme proteins and phospholipids within microsomal membranes was studied by analyzing membrane composition after treatment with proteases and phospholipases. Upon trypsin treatment of closed microsomal vesicles, NADH- and NADPH-cytochrome c reductases as well as cytochrome b5 were solubilized or inactivated, while cytochrome P-450 was partially inactivated. When microsomes were exposed to a concentration of deoxycholate which makes them permeable to macromolecules but does not disrupt the membrane, the detergent alone was sufficient to release four enzymes: nucleoside diphosphatase, esterase, beta-glucuronidase, and a portion of the DT-diaphorase. Introduction of trypsin into the vesicle lumen inactivated glucose-6-phosphatase completely and cytochrome P-450 partially. The rest of this cytochrome, ATPase, AMPase, UDP-glucuronyltransferase, and the remaining 50% of DT-diaphorase activity were not affected by proteolysis from either side of the membrane. Phospholipase A treatment of intact microsomes in the presence of albumin hydrolyzed all of the phosphatidylethanolamine, phosphatidylserine, and 55% of the phosphatidylcholine. From this observation, it was concluded that these lipids are localized in the outer half of the bilayer of the microsomal membrane; Phosphatidylinositol, 45% of the phosphatidylcholine, and sphingomyelin are tentatively assigned to the inner half of this bilayer. It appears that the various enzyme proteins and phospholipids of the microsomal membrane display an asymmetric distribution in the transverse plane.     

Lipid asymmetry induced by transmembrane pH gradients in large unilamellar vesicles

We have investigated the influence of transmembrane pH gradients across large unilamellar vesicle membranes on the transbilayer distributions of simple lipids with weak base and weak acid characteristics. Trinitrobenzenesulfonic acid labeling results consistent with a rapid and complete migration of stearylamine and sphingosine to the inner monolayer of the large unilamellar vesicles are observed when the large unilamellar vesicles' interior is acidic. Alternatively, when the vesicle interior is basic, oleic and stearic acid cannot be removed by external bovine serum albumin, indicating a localization in the inner monolayer. Moreover, effects corresponding to the decrease in external surface charge predicted upon the migration of stearylamine or stearic acid to the inner monolayer are readily detected employing ion exchange chromatography. These results are consistent with transbilayer distributions of these agents dictated by a Henderson-Hasselbach equilibrium. The possible implications for metabolic regulation by pH gradients, as well as factors giving rise to phospholipid transbilayer asymmetry, are discussed.     

Phospholipid asymmetry in large unilamellar vesicles induced by transmembrane pH gradients

The influence of membrane pH gradients on the transbilayer distribution of some common phospholipids has been investigated. We demonstrate that the transbilayer equilibrium of the acidic phospholipids egg phosphatidylglycerol (EPG) and egg phosphatidic acid (EPA) can be manipulated by membrane proton gradients, whereas phosphatidylethanolamine, a zwitterionic phospholipid, remains equally distributed between the inner and outer monolayers of large unilamellar vesicles (LUVs). Asymmetry of EPG is examined in detail and demonstrated by employing three independent techniques: ion-exchange chromatography, 13C NMR, and periodic acid oxidation of the (exterior) EPG headgroup. In the absence of a transmembrane pH gradient (delta pH) EPG is equally distributed between the outer and inner monolayers of LUVs. When vesicles composed of either egg phosphatidylcholine (EPC) or DOPC together with 5 mol % EPG are prepared with a transmembrane delta pH (inside basic, outside acidic), EPG equilibrates across the bilayer until 80-90% of the EPG is located in the inner monolayer. Reversing the pH gradient (inside acidic, outside basic) results in the opposite asymmetry. The rate at which EPG equilibrates across the membrane is temperature dependent. These observations are consistent with a mechanism in which the protonated (neutral) species of EPG is able to traverse the bilayer. Under these circumstances EPG would be expected to equilibrate across the bilayer in a manner that reflects the transmembrane proton gradient. A similar mechanism has been demonstrated to apply to simple lipids that exhibit weak acid or base characteristics [Hope, M. J., & Cullis, P. R. (1987) J. Biol. Chem 262, 4360-4366]     

Genetic toggle switch controlled by bacterial growth rate

Background

In favorable conditions bacterial doubling time is less than 20 min, shorter than DNA replication time. In E. coli a single round of genome replication lasts about 40 min and it must be accomplished about 20 min before cell division. To achieve such fast growth rates bacteria perform multiple replication rounds simultaneously. As a result, when the division time is as short as 20 min E. coli has about 8 copies of origin of replication (ori) and the average copy number of the genes situated close to ori can be 4 times larger than those near the terminus of replication (ter). It implies that shortening of cell cycle may influence dynamics of regulatory pathways involving genes placed at distant loci.

Results

We analyze this effect in a model of a genetic toggle switch, i.e. a system of two mutually repressing genes, one localized in the vicinity of ori and the other localized in the vicinity of ter. Using a stochastic model that accounts for cell growth and divisions we demonstrate that shortening of the cell cycle can induce switching of the toggle to the state in which expression of the gene placed near ter is suppressed. The toggle bistability causes that the ratio of expression of the competing genes changes more than two orders of magnitude for a two-fold change of the doubling time. The increasing stability of the two toggle states enhances system sensitivity but also its reaction time.

Conclusions

By fusing the competing genes with fluorescent tags this mechanism could be tested and employed to create an indicator of the doubling time. By manipulating copy numbers of the competing genes and locus of the gene situated near ter, one can obtain equal average expression of both genes for any doubling time T between 20 and 120 min. Such a toggle would accurately report departures of the doubling time from T.

     

Fission of membrane nanotube induced by osmotic pressure

The final stage of endocytosis is fission of a thin membrane neck, or nanotube (NT), connecting cell membrane with a forming vesicle. We studied this process using a model system consisting of NT pulled out from a flat bilayer lipid membrane. Fission of NT was induced by an increase of osmotic pressure created by local application of a concentrated salt solution in the vicinity of NT. Superfusion of NT with distilled water instead of the concentrated salt solution led to the NT expansion. This observation demonstrates the reversibility of the NT expansion-compression process under the osmotic pressure. The overall picture of fission is similar to that described earlier for the NT fission with participation of dynamin GTPase. In both cases, in order for fission to occur, it is necessary to compress the NT to a critical radius. The critical radius estimated for the osmotic pressure-induced fission exceeds the value obtained for the fission occurring in the presence of the protein. Fission under osmotic pressure, akin the dynamin-promoted fission, proceeds without leaky defects.     

Control of the transmembrane phospholipid distribution in eukaryotic cells by aminophospholipid translocase

The aminophospholipid translocase is a plasma membrane Mg2(+)-ATPase which selectively pumps the aminophospholipids (phosphatidylserine and phosphatidylethanolamine) from the outer to the inner monolayer in eukaryotic cells and is predominantly responsible for the asymmetric phospholipid distribution of the plasma membrane. Similar ATP-dependent transport of phospholipid takes place in some organelles such as chromaffin granules. On the other hand, the phospholipid flippase of rat liver endoplasmic reticulum does not require ATP and has a low lipid specificity. The biological implications of these phospholipid flippases are discussed.     

Changes in the phospholipid composition and phospholipid asymmetry of ram sperm plasma membranes after cryopreservation

The changes in the phospholipid composition of spermatozoa plasma membranes after freezing were determined by thin-layer chromatography. The results showed an augmentation of the diphosphatidylglycerol and a diminution of phosphatidylglycerol, phosphatidylserine, and phosphatidylethanolamine in sperm plasma membranes after freezing. In intact sperm cells we observed an elevation of the sphingomyelin and phosphatidylinositol levels and a diminution of the phosphatidylethanolamine and diphosphatidylglycerol levels. The effect of freezing on the phospholipid distribution between the inner and outer monolayers of the plasma membrane was also studied using exogenous phospholipases and trinitrobenzene sulfonate. The most important change we observed after freezing, was the translocation of diphosphatidylglycerol from the inner to the outer monolayer of the plasma membrane.     

Comparison of the effects of surface tension and osmotic pressure on the interfacial hydration of a fluid phospholipid bilayer

The effects of three so-called kosmotropic solutes, namely, betaine, sucrose, and choline chloride on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine large unilamellar vesicles, were studied by measuring the generalized polarization (GP) for the fluorescence emission of the membrane partitioning probe Laurdan. The latter has been shown to be sensitive to the depth of water penetration into phospholipid bilayers. At equal osmotic pressures the three solutes produced different increments in GP, with a qualitative positive correlation. However, the increments in GP correlated also quantitatively with the increase of air-water surface tension caused by the three kosmotropes. Our findings suggest surface tension to determine the impact of these solutes on the lateral packing of the lipid bilayer. Based on the changes in area/lipid at different surface tensions, the equilibrium lateral pressure for a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer at 25 degrees C was estimated to be approximately 34 mN/m.     

Phase transitions as a function of osmotic pressure in Saccharomyces cerevisiae whole cells, membrane extracts and phospholipid mixtures

Fourier Transform Infrared spectroscopy (FTIR) was used to determine the phase transition temperature of whole Saccharomyces cerevisiae W303-1 A cells as a function of Aw in binary water-glycerol media. A phase transition occurred at 12 degrees C in water, at 16.5 degrees C at Aw=0.75, and at 19.5 degrees C at Aw=0.65. The temperature ranges over which transition occurred increased with decreasing Aw. A total lipid extract of the plasma membranes isolated from S. cerevisiae cells was also studied, with a phase transition temperature determined at 20 degrees C in pure water and at 27 degrees C in binary water-glycerol solutions for both Aw levels tested. The pure phospholipids dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) and three binary mixtures of these phospholipids (percentage molar mixtures of DMPC/DMPE of 90.5/9.5, 74.8/25.2, and 39.7/60.3) were studied. For DMPC, there was no influence of Aw on the phase transition temperature (always 23 degrees C). On the other hand, the phase transition temperature of DMPE increased with decreasing Aw for the three aqueous solutions tested (glycerol, sorbitol and sucrose), from 48 degrees C in water, to 64 degrees C for a solution at Aw=0.67. For the DMPC/DMPE mixtures, transitions were found intermediate between those of the two phospholipids, and a cooperative state was observed between species at the gel and at the fluid phases.     

Erythritol production by controlling osmotic pressure in Trigonopsis variabilis

Specific erythritol production rate by Trigonopsis variabilis increased from 0.09 to 0.19 g/g-day by varying the osmotic pressure from 1.3 to 3.9 kPa in glucose medium, but the specific growth rate decreased from 0.28 to 0.13 h -1 . Therefore, osmotic pressure was adjusted to 1.4 kPa during growth phase and to 3.7 kPa during production phase in a two-stage fermentation. Erythritol reached 46 g/ l in such a system and was twice that obtained in one-stage fermentation.     

Proteins induced by high osmotic pressure in Escherichia coli

Abstract The protein composition of Escherichia coli grown under conditions of high and low osmotic pressure and following shifts from one condition to the other was examined by two-dimensional gel electrophoresis. Upon shift to high osmotic pressure there is a rapid induction of a group of proteins whose synthesis persists during prolonged growth at elevated osmotic pressure. An osmotic down-shift results in the repression of these proteins. Neither shift induced the heat shock regulon.     

Functional and pathological significance of phospholipid asymmetry in erythrocyte membranes

The normal asymmetric distribution of phospholipids in the plasma membrane is perturbed in erythrocytes from patients with chronic myelogenous leukemia. Since experimentally-produced lipid-symmetric erythrocytes are more interactive with cells of the reticuloendothelial system than are their lipid-asymmetric counterparts, the biological recognition of chronic myelogenous leukemia erythrocytes by the reticuloendothelial system was examined. With one exception, all erythrocyte samples from patients with chronic/benign chronic myelogenous leukemia were more adherent to endothelial cells and more readily phagocytosed by macrophagesin vitro than were normal erythrocytes. Thus, these naturally occurring pathological erythrocytes display the same dysfunctional intercellular interactions as the laboratory models.     

Mechanistic formalism for membrane transport generated by osmotic and mechanical pressure

Since the physical interpretation of practical Kedem-Katchalsky (KK) equations is not clear, we consider an alternative, mechanistic approach to membrane transport generated by osmotic and hydraulic pressure. We study a porous membrane with randomly distributed pore sizes (radii). We postulate that reflection coefficient (sigma p) of a single pore may equal 1 or 0. From this postulate we derive new (mechanistic) transport equations. Their advantage is in clear physical interpretation and since we show they are equivalent to the KK equations, the interpretation of the latter became clearer as well. Henceforth the equations allow clearer and more detailed interpretation of results concerning membrane mass transport. This is especially important from the point of view of biophysical studies on permeation processes across biological membranes, cell membranes including.