The interaction of saccharides with lipid bilayer vesicles: stabilization during freeze-thawing and freeze-drying

The fusion of small unilamellar vesicles of phosphatidylcholines during freeze-thawing and freeze-drying/rehydration, and the suppression of fusion under these conditions by various saccharides, was investigated by gel filtration on Sepharose 4B, quasielastic light scattering, high-resolution 1H-NMR, ESR spin labeling, and differential scanning calorimetry. Freeze-thawing and freeze-drying of aqueous small unilamellar vesicle suspensions in the presence of sufficient sucrose had no significant effect on the average size and size distribution of small unilamellar vesicles. In the presence of sucrose the structural integrity and the permeability properties of the phosphatidylcholine bilayers were retained during freeze-thawing and freeze-drying. A comparison of the stabilizing effect of sucrose with that of trehalose and glucose showed that the stabilization is not sugar-specific but is a general property of saccharides. The fraction of small unilamellar vesicles recovered after freeze-thawing depended on the saccharide/phosphatidylcholine molar ratio. The mechanism of the cryoprotective effect involves binding of the sugar to the phospholipid polar group, probably through hydrogen bonding.     

Binding of peroxiredoxin 6 to substrate determines differential phospholipid hydroperoxide peroxidase and phospholipase A2 activities

Peroxiredoxin 6 (Prdx6) differs from other mammalian peroxiredoxins both in its ability to reduce phospholipid hydroperoxides at neutral pH and in having phospholipase A₂ (PLA₂) activity that is maximal at acidic pH. We previously showed an active site C47 for peroxidase activity and a catalytic triad S32-H26-D140 necessary for binding of phospholipid and PLA₂ activity. This study evaluated binding of reduced and oxidized phospholipid hydroperoxide to Prdx6 at cytosolic pH. Incubation of recombinant Prdx6 with 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine hydroperoxide (PLPCOOH) resulted in peroxidase activity, cys47 oxidation as detected with Prdx6-SO₂₍₃₎ antibody, and a marked shift in the Prdx6 melting temperature by circular dichroism analysis indicating that PLPCOOH is a specific substrate for Prdx6. Preferential Prdx6 binding to oxidized liposomes was detected by changes in DNS-PE or bis-Pyr fluorescence and by ultrafiltration. Site-specific mutation of S32 or H26 in Prdx6 abolished binding while D140 mutation had no effect. Treatment of A549 cells with peroxides led to lipid peroxidation and translocation of Prdx6 from the cytosol to the cell membrane. Thus, the pH specificity for the two enzymatic activities of Prdx6 can be explained by the differential binding kinetics of the protein; Prdx6 binds to reduced phospholipid at acidic pH but at cytosolic pH binds only phospholipid that is oxidized compatible with a role for Prdx6 in the repair of peroxidized cell membranes.     

Alterations in the phospholipid composition of Escherichia coli B during growth at different temperatures

The major phospholipid classes of Escherichia coli B, phosphatidyl ethanolamine, cardiolipin, and phosphatidyl glycerol, were quantitated at different stages of the growth cycle. The organisms were incubated at both 27 and 37 C. Significant differences were observed both in the amounts of total lipid phosphorus per gram (dry weight) of cells and in the relative percentages of the individual phospholipids. At 37 C the total amount of lipid phosphorus decreased significantly throughout the growth cycle. However, at 27 C total lipid phosphorus accumulated. The patterns of the three major phospholipid classes of Escherichia coli exhibited complex quantitative changes. In addition, some evidence based on glycerol to phosphate molar ratios indicated that phosphatidyl glycerolphosphate replaced phosphatidyl glycerol during the late growth stages of E. coli B when grown at 27 C. A comparative analysis of phospholipid and fatty acid patterns led to a hypothesis attempting to explain some reported variations in the lipid composition of E. coli under different conditions of growth.     

Phospholipid Composition of Bacillus subtilis

Bacillus subtilis contained at least five phospholipids, four of which have been isolated and identified as a polyglycerol phospholipid, probably cardiolipin, phosphatidylglycerol, phosphatidylethanolamine, and lysylphosphatidylglycerol. Further purification of the latter phosphoglyceride was obtained by high-voltage electrophoresis, and it was shown that this treatment removed amino acid-containing, nonlipidic material from the phosphoglyceride. This associated material, which is not covalently linked to the lipid, gave rise to minor amounts of a number of amino acids, other than lysine, in acid hydrolysates of the lysylphosphatidylglycerol. The phospholipid composition of B. subtilis appeared to depend on the growth conditions. Addition of glucose to the medium lowered the pH during growth; this was accompanied by an increase in the amount of lysylphosphatidylglycerol and a decrease in the phosphatidylglycerol content, when compared with growth at neutral pH. The amount of the other phospholipids and the total amount of phospholipid remained constant under the different conditions. The shape and the osmotic susceptibility of the protoplasts of this organism appeared to depend on the growth conditions. Cells harvested from a neutral growth medium gave spherical protoplasts which lysed rapidly, whereas cells grown in an acidic medium maintained their rod-shaped form to a great extent after the cell wall had been removed, even after being suspended in a hypotonic medium. The latter observation suggests the presence of a more rigid membranous structure in cells which have been exposed to a low environmental pH during growth.     

Cholesterol metabolism by Mycobacterium.

Cholesterol metabolism by Mycobacterium species ATCC Number 19652 was studied in defined media. Whole cells were found to take up 91% of the total cholesterol when incubated five days at 34 degrees C in media of pH 6.8-7.4. Uptake of cholesterol by whole cells could be significantly inhibited by 2,4-dinitrophenol and dicyclohexylcarbodiimide. Growth media supernates as well as isolated microbial cell walls were found to contain cholesterol hydrolysing activity. This activity was extractable by Triton X-100 and appeared to have a molecular weight of approximately 100-200,000.     

Effects of hyperbaric oxygen on the growth and properties of Pseudomonas aeruginosa

Growth of Pseudomonas aeruginosa in 2 atmospheres absolute of 100% oxygen at 37 degrees C produced two types of abnormal colonies--stunted, rough colonies, termed dwarfs, and large, domed, mucoid colonies, termed giants. The occurrence of these variants depended upon the partial pressure of oxygen and the inoculum size. Subculture of dwarf or giant colonies produced a mixture of both colony types after incubation in hyperbaric oxygen, and colonies of normal appearance after incubation in air. Electron micrographs of ultrathin sections showed that cells from dwarf colonies had a more clearly defined envelope region than cells from normal colonies. Giant colony and normal colony-derived cells were of similar appearance. Whole cells from giant colonies contained more carbohydrate, readily extractable lipid, neutral lipid and free fatty acid than cells from normal colonies; the two cell types showed similar contents of 2-keto,3-deoxyoctonic acid and total phospholipid, but different proportions of individual phospholipids. Cells from dwarf, giant and normal (air-grown) colonies were incubated in air on nutrient agar containing either polymyxin, tetracycline or phenoxyethanol. Relative to cells from normal colonies, cells from dwarf colonies showed enhanced resistance to all three agents and cells from giant colonies showed enhanced resistance to polymyxin and tetracycline only. The resistance of cells from variant colonies was lost following a single subculture in air in the absence of antibacterial agents. It was concluded that the envelopes of cells from dwarf and giant colonies differed both from each other and from those of normal cells. These differences, and the formation of variant colonies, appeared to result from bacterial adaptation to hyperbaric oxygen rather than from mutation.     

Sulfur-related air pollutants induce the generation of platelet-activating factor, 5-lipoxygenase- and cyclooxygenase-products in canine alveolar macrophages via activation of phospholipases A2

Recent studies have shown that long-term in vivo exposure of dogs to neutral sulfur(IV)/sulfite aerosols induces mild inflammatory reactions, whereas the combination of neutral sulfite with acidic sulfur(VI)/sulfate aerosols evokes less pronounced effects. To understand underlying mechanisms, we studied in vitro the role of lipid mediators in the responses of alveolar macrophages (AMs) to sulfur-related compounds under neutral (pH 7) or moderate acidic (pH 6) conditions. Canine AMs incubated with sulfite at pH 7 released threefold higher amounts of platelet-activating factor than control (P < 0.005). Generation of arachidonic acid, leukotriene B4, 5-hydroxy-eicosatetraenoic acid, prostaglandin E2, thromboxane B2 and 12-hydroxyheptadecatrienoic acid increased twofold (P < 0.0005). However, these metabolites remained unchanged following incubation of AMs with sulfite at pH 6 or with sulfate at pH 7 or pH 6. Mediator release by sulfite-treated AMs at pH 7 stimulated respiratory burst activity of neutrophils. Inhibition of MAPK pathway by PD 98059, of cytosolic (cPLA2) and secretory phospholipases A2 by AACOCF3 and thioetheramide-PC, respectively, reduced sulfite-induced eicosanoid formation in AMs. Sulfite activated cPLA2 activity twofold at pH 7. This mechanism of sulfite-stimulated responses in phospholipid metabolism predicts that chronic exposure to sulfur(IV)/sulfite is associated with a considerable health risk.     

Variation in Lipid Content of Strains of Histoplasma capsulatum Exhibiting Different Virulence Properties for Mice

Nielsen, H. S., Jr. (Duke University Medical Center, Durham, N.C.). Variation in lipid content of strains of Histoplasma capsulatum exhibiting different virulence properties for mice. J. Bacteriol. 91:273-277. 1966.-Lipid content and virulence were studied in six isolates of Histoplasma capsulatum in an attempt to determine whether or not the two factors could be correlated in this fungus. Virulence was evaluated by injecting dba line 1 male mice intracerebrally with 2.8 x 10(4) infective yeast-phase units and recording organ involvement and spontaneous deaths occurring in a 20-day period. Yeast cells were extracted with mixtures of ethyl alcohol-diethyl ether (3:1, v/v), and the total extractable lipid, as determined by solubility in petroleum ether, was separated into acetone-soluble and phospholipid fractions by acetone precipitation. Neutral lipids were measured directly by weighing, whereas total phospholipids were calculated after the colorimetric determination of phosphorus. The mixed phosphatides of two isolates, differing in virulence, were separated into five fractions by use of a column of silicic acid and Hyflo Super-Cel. In the six isolates studied, neither total extractable lipid, acetone-soluble lipid, nor phospholipid showed a quantitative correlation with virulence. Phosphatidylserine, cephalin, phosphoinositides, and sphingolipids were present in essentially the same amounts in the two strains investigated; however, a lecithin fraction was absent in the less virulent form. These data suggest that the quantity of phosphatidylcholine demonstrated for a given isolate of H. capsulatum may provide some insight as to its virulence, although such a relationship is lacking for total lipid, the acetone-soluble fraction, and the combined phospholipids of yeast-phase growth.     

Interaction of added amphiphilic lipids with the membrane of intact human erythrocytes to induce change in the cell shape

Addition of an appropriate amount of amphiphilic lipid, such as fatty acid, lysophospholipid and medium-chain phospholipid, into a suspension of human erythrocytes (pH 7.4) at 37 degree C resulted in their incorporation into the membrane and induction of a cell shape change of crenation (echinocyte-spherocyte) type without causing hemolysis. The extent of the shape change was dependent on the amount of the lipid incorporated and the crenation disappeared on removing the incorporated molecules from the membrane. The crenation induced by acidic lipids was further altered drastically by resuspending the treated cells in media of pH 6, 7, and 8, whereas that induced by choline-phospholipid or -lysophospholipid was not so pH-dependent. Based on these results, the mechanism of this shape change is discussed.     

Effect of lipid composition on the stability of cellular membranes during freeze-thawing of Lactobacillus acidophilus grown at different temperatures

Lactobacillus acidophilus CRL 640 grown at the optimal temperature of 37 degrees C (M37) appeared more sensitive to freeze-thawing than when it was grown at 25 degrees C (M25). In the first case, 87% of the cells died, in contrast to 33% for cells grown at 25 degrees C. All the surviving M37 cells showed sensitivity to NaCl. However, among the surviving M25 cells, only 85% were sensitive to NaCl. The rest of the cells were considered uninjured. Freeze-thawing in cells grown at 25 degrees C showed a liberation of nucleic acids and proteins. However, the leakage was higher in M37 cells after freeze-thawing. The greater fraction of damaged cells were observed in M25 culture after freeze-thawing. A relative increase of 81% in cardiolipid (CL), with respect to total phospholipids and 72% triglycosyldiglyceride (TGDG) with respect to the total glycolipids was observed in M37. In addition, a decrease of palmitoyl (C16:0), oleoyl (C18:0) fatty acids at CL, phosphatidylglycerol (PG), and diglicosyldiglyceride (DGDG) fractions and the increase of C19 cyc and C18:0, 10-OH fatty acids in neutral lipid, and CL fractions was also apparent. In M25 cells, the concentration of DGDG and PG was higher than in M37 cells. The difference in cryotolerance between the frozen cultures emphasizes the importance of selecting appropriate conditions of growth of microorganisms for use as dietary adjuncts.     

Activity and properties of CTP: cholinephosphate cytidylyltransferase in adult and fetal rat lung.

Cholinephosphate cytidylyltransferase (CTP : cholinephosphate cytidylyltransferase, EC 2.7.7.15) is located in both the microsomal and supernatant fractions of adult lung when the tissue is homogenized in 0.145 M NaCl. The activity is located predominantly in the supernatant fraction in fetal lung. Cholinephosphate cytidylyltransferase in the supernatant from fetal lung is stimulated 4- to 6-fold by the additions of total lung lipid. Serine phosphoglycerides and inositol phosphoglycerides specifically caused stimulation whereas choline phosphoglycerides and ethanolamine phosphoglycerides produced no stimulation. Lysophosphatidylcholine cause some stimulation, but only at high concentrations. A number of detergents were investigated. All produced inhibition except for the ampholytic detergent, miranol H2M which was not inhibitory. None of the detergents produced any stimulation of activity. Cytidylyltransferase activity in fetal lung when assayed in the absence of lipid is about 25% of the adult. The activity when assayed in the presence of lipid is equal or slightly higher than adult levels. The activity, measured without added phospholipid, increases 5- to 6-fold within 12 h after birth, to values higher than in the adult. The activity, measured in the presence of phospholipid, increased almost linearly from -2 day until +1 day. There is an inverse relationship between the concentration of phospholipid in the fetal lung supernatant and the degree of lipid stimulation. Chromatographic experiments with Biogel A 1.5 columns have shown that cytidylyltransferase can exist in two molecular sizes, a small molecular size that requires phospholipid for activity, and a larger molecular weight species which does not require the addition of phospholipid for activity. Fetal lung has a higher proportion of the low molecular weight form than adult lung. The small molecular weight species can be converted to the larger molecular weight form by the addition of phospholipids.     

Effect of varying static and changing moisture levels during incubation on extractability of soil phosphate

Summary A sandy loam (pH 6.5) was incubated at 28°C at static moisture levels, ranging from 10 per cent saturation to 133 per cent saturation (waterlogging), for 6 and 12 weeks; other samples covering the same moisture range were first incubated for 6 weeks, and after changing all moisture levels to 50 per cent saturation were incubated for a further 6 weeks.With increasing static soil moisture level during incubation there was a slight reduction in Morgan-extractable phosphate up to 70 per cent saturation, but thereafter, due to anaerobic effects, there were considerable increases in extractable phosphate with increasing moisture level.With changing moisture level during incubation the effects of anaerobiosis became apparent where original moisture level was greater than 50 per cent saturation; extractable phosphate was reduced to levels lower than those occurring where the soil was maintained continuously at 50 per cent saturation. The extent of reduction in extractable phosphate increased with original soil saturation level.     

Phospholipid composition and phospholipase A activity of Neisseria gonorrhoeae.

Exponential-phase cells of Neisseria gonorrhaeae 2686 were examined for phospholipid composition and for membrane-associated phospholipase A activity. When cells were harvested by centrifugation, washed, and lyophilized before extraction, approximately 74% of the total phospholipid was phosphatidylethanolamine, 18% was phosphatidylglycerol, 2% was cardiolipin, and 10% was lysophosphatidylethanolamine. However, when cells still suspended in growth medium were extracted, the amount of lysophosphatidylethanolamine decreased to approximately 1% of the phospholipid composition. This suggests that a gonococcal phospholipase A may be activated by conditions encountered during centrifugation and/or lyophilization of cells preceding extraction. Phospholipase A activity associated with cell membranes was assayed by measuring the conversion of tritiated phosphatidylethanolamine to lysophosphatidylethanolamine. Optimal activity was demonstrated in 10% methanol at pH 8.0 to 8.5, in the presence of calcium ions. The activity was both detergent sensitive and thermolabile. Comparisons of gonococcal colony types 1 and 4 showed no significant differences between the two types with respect to either phospholipid content or phospholipase A activity.     

Inhibitory effects of basic or neutral phospholipid on acidic phospholipid-mediated dissociation of adenine nucleotide bound to DnaA protein,the initiator of chromosomal DNA replication

DnaA protein activity, the initiator of chromosomal DNA replication in bacteria, is regulated by acidic phospholipids such as phosphatidylglycerol (PG) or cardiolipin (CL) via facilitation of the exchange reaction of bound adenine nucleotide. Total lipid isolated from exponentially growing Staphylococcus aureus cells facilitated the release of ATP bound to S. aureus DnaA protein, whereas that from stationary phase cells was inert. Fractionation of total lipid from stationary phase cells revealed that the basic phospholipid, lysylphosphatidylglycerol (LPG), inhibited PG- or CL-facilitated release of ATP from DnaA protein. There was an increase in LPG concentration during the stationary phase. A fraction of the total lipid from stationary phase cells of an integrational deletion mprF mutant, in which LPG was lost, facilitated the release of ATP from DnaA protein. A zwitterionic phospholipid, phosphatidylethanolamine, also inhibited PG-facilitated ATP release. These results indicate that interaction of DnaA protein with acidic phospholipids might be regulated by changes in the phospholipid composition of the cell membrane at different growth stages. In addition, the mprF mutant exhibited an increased amount of origin per cell in vivo, suggesting that LPG is involved in regulating the cell cycle event(s).     

Lipid requirement of the membrane sodium-plus-potassium ion-dependent adenosine triphosphatase system.

The dependence of the (Na-++K-+)-dependent ATPase (adenosine triphosphatase) (EC 3.6.1.3) on lipid has been examined in a number of different ways, with the use of various preparations from kidney tissue. The main findings were as follows. (1) The ATPase activities of the preparations examined were closely correlated with their total phospholipid content. (2) Extraction of the ATPase with deoxycholate or Lubrol W, combined with suitable salt-fractionation and washing procedures, removed phospholipid, cholesterol and enzymic activity in parallel; but activity was completely lost before all lipid had been removed. (3) The loss of activity could not be attributed to inhibition by residual detergent. (4) No selective removal of any particular phospholipid class by detergent could be detected. (5) Consistent reactivation of the Lubrol-extracted enzymes was obtained by adding dispersions of exogenous phospholipid, but only some, bearing a net negative charge, such as phosphatidylserine and phosphatidylglycerol, were effective. (6) The degree of reactivation was correlated with the amount of residual activity remaining after lipid depletion. (7) Partial purification of the ATPase, giving a 50-fold increase in specific activity, was not accompanied by selective enhancement of any particular class of phospholipid. We conclude that although the ATPase is dependent on phospholipid, only the reactivation results provide evidence for specificity.     

Studies on the damage to Escherichia coli cell membrane caused by different rates of freeze-thawing

Freeze-thawing of Escherichia coli cells caused a release of cell membrane components such as protein, phospholipids and lipopolysaccharides. A greater amount of release and a lesser extent of cell survival were seen in slow freeze-thawing than in rapid freeze-thawing. Several dehydrogenases in the cells were also freed. The mode of release was also dependent on the rate of freeze-thawing.The materials released by slow freeze-thawing were found to be mostly composed of outer membrane components, whereas the materials released by rapid freeze-thawing contained cytoplasmic as well as outer membrane components. The chemical composition of these fragments differed significantly from that of the original membranes. The relative content of cytoplasmic membrane-bound enzymes in these fragments also differed from that of the cytoplasmic membrane.The fragmentation was assumed to have resulted mainly from the crystallization of external water. In slow freeze-thawing, it was considered that the phase separation of the membrane phospholipid bilayer increased the possibility of outer membrane fragmentation. Rapid freeze-thawing caused cytoplasmic membrane damage to the cells as well as to the outer membrane. In rapid freeze-thawing, the effect of phase separation appeared to be small because of rapid passage through the transition temperatures.The presence of 10% glycerol completely inhibited the release of cellular materials and enzymes. Cell survival was maintained at a high level in the glycerol-treated samples whether freeze-thawed slowly or rapidly.     

Maintenance of quaternary structure in the frozen state stabilizes lactate dehydrogenase during freeze-drying

Sugars inhibit protein unfolding during the drying step of lyophilization by replacing hydrogen bonds to the protein lost upon removal of water. In many cases, polymers fail to inhibit dehydration-induced damage to proteins because steric hindrance prevents effective hydrogen bonding of the polymer to the protein's surface. However, in certain cases, polymers have been shown to stabilize multimeric enzymes during lyophilization. Here we test the hypothesis that this protection is due to inhibition of dissociation into subunits during freezing. To test this hypothesis, as a model system we used mixtures of lactate dehydrogenase isozymes that form electrophoretically distinguishable hybrid tetramers during reversible dissociation. We examined hybridization and recovery of catalytic activity during freeze-thawing and freeze-drying in the presence of polymers (dextran, Ficoll, and polyethylene glycol), sugars (sucrose, trehalose, glucose), and surfactants (Tween 80, Brij 35, hydroxy-propyl beta-cyclodextrin). The surfactants did not protect LDH during freeze-thawing or freeze-drying. Rather, in the presence of Brij 35, enhanced damage was seen during both freeze-thawing and freeze-drying, and the presence of Tween 80 exacerbated loss of active protein during freeze-drying. Polymers and sugars prevented dissociation of LDH during the freezing step of lyophilization, resulting in greater recovery of enzyme activity after lyophilization and rehydration. This beneficial effect was observed even in systems that do not form glassy solids during freezing and drying. We suggest that stabilization during drying results in part from greater inherent stability of the assembled holoenzyme relative to that of the dissociated monomers. Polymers inhibit freezing-induced dissociation thermodynamically because they are preferentially excluded from the surface of proteins, which increases the free energy of dissociation and denaturation.     

Lipid Content of Antibiotic-Resistant and -Sensitive Strains of Serratia marcescens

The lipid content of antibiotic-resistant, nonpigmented strain (Bizio) and antibiotic-sensitive, pigmented strain (08) of Serratia marcescens was studied. The resistant strain contains at least three times more total extractable lipid and phospholipid than the sensitive strain. Lysophosphatidylethanolamine, phosphatidylserine, lecithin, phosphatidylglycerol, phosphatidylethanolamine, and polyglycerolphosphatide were identified in the phospholipid fractions of both strains.     

Phospholipids from Bacillus stearothermophilus

The lipids of Bacillus stearothermophilus strain 2184 were extracted with chloroform-methanol and separated into neutral lipid and three phospholipid fractions by chromatography on silicic acid columns. The phospholipids were identified by specific staining reactions on silicic acid-impregnated paper, by chromatography of alkaline and acid hydrolysis products, and by determination of acyl ester:glycerol:nitrogen:phosphorus molar ratios. The total extractable lipid was 8% of the dry weight of whole cells and consisted of 30 to 40% neutral lipid and 60 to 70% phospholipid. The phospholipid consisted of diphosphatidyl glycerol (23 to 42%), phosphatidyl glycerol (22 to 39%), and phosphatidyl ethanolamine (21 to 32%). The concentrations of diphosphatidyl glycerol and phosphatidyl glycerol were lower in 2-hr cells than in 4- and 8-hr cells. Whole cells were fractionated by sonic treatment and differential centrifugation. The total lipid content, expressed in per cent of dry weight of each fraction was: whole protoplasts, 10%; membrane fraction, 18%; 30,000 x g particulate fraction, 22%; and 105,000 x g particulate fraction, 26%. The relative phospholipid concentrations in each fraction were about the same. As had been previously reported, none of the phospholipid was stable to alkaline hydrolysis.     

Preservation of integrity of the inner mitochondrial membrane after freeze-thawing and freeze-drying

The results of this paper illustrate that trehalose partially preserves inner mitochondrial membrane integrity after freeze-thawing and freeze-drying with subsequent rehydration in water. The 2,4-dinitrophenol stimulation of ATPase activity was used as a criterion for membrane integrity. The results show that ATPase activity of lyophilized-rehydrated mitochondria was stimulated up to two to three times.