Infrared spectroscopic study of the gel to liquid-crystal phase transition in live Acholeplasma laidlawii cells

The temperature dependences of the infrared spectra of deuterium-labeled plasma membranes of live Acholeplasma laidlawii B cells and of the isolated plasma membranes demonstrate that the profiles of the gel to liquid-crystal phase transitions are very different. At temperatures within the range of the phase transition, the live mycoplasma is able to keep the "fluidity" of its plasma membrane at a much higher value than that of the isolated plasma membrane at the same temperature. The difference is particularly pronounced at and around the temperature of growth. Live Acholeplasma laidlawii, grown at 37 degrees C on a fatty acid depleted medium supplemented with myristic acid (C14:0), pentadecanoic acid (C15:0), or palmitic acid (C16:0), are highly "fluid"; i.e., at the temperature of growth, the fractional population of the liquid-crystalline phase is 95-100% at 37 degrees C, whereas in the case of the isolated plasma membranes the fractional population of the liquid-crystalline phase at 37 degrees C is only 58% (C14:0), 36% (C15:0), or 38% (C16:0).     

Binding of lectins to membranes of mycoplasmas from aging cultures

The binding of iodinated concanavalin A (Con A) and Ricinus communis agglutinin (RCA) to intact cells and isolated membranes of Acholeplasma laidlawii, Mycoplasma hominis and Mycoplasma capricolum decreased with the progression of the culture from the mid- to the late-logarithmic phase of growth. The binding of the lectins to Acholeplasma laidlawii membranes had no significant effect on membrane fluidity, as assessed by electron-paramagnetic resonance spectroscopy of spin-labelled fatty acids, and had no effect on several membrane-associated enzymic activities. Temperature affected the binding of Con A and RCA in an opposite manner: the binding of Con A increased, whereas that of RCA decreased, on raising the temperature from 4 degrees C to 37 degrees C. No significant difference in lectin binding was found between oleate- and elaidate-enriched membranes at low temperatures where the former was in the liquid-crystalline state and the latter in the gel state, suggesting that membranes fluidity does not influence the binding of Con A and RCA to Acholeplasma laidlawii membranes.     

Studies on Acholeplasma laidlawii grown on branched-chain fatty acids

Acholeplasma laidlawii B was grown on the branched-chain fatty acids, 14-methylpentadecanoic acid and 14-methylhexadecanoic acid, and the straight-chain palmitic acid. The incorporation of the branched-chain fatty acids was very effective; more than 90% of the fatty acids of the lipids of this organism consisted of the branched-chain constituents. A somewhat smaller amount (81%) was found in the cells grown with palmitic acid. Differential scanning calorimetry of the isolated membranes showed that distinct lipid phase transitions occurred in between 15 and 31 °C for the 14-methylpentadecanoic acid, 11 and 29 °C for the 14-methylhexadecanoic acid, and 14 and 36 °C for the palmitic acid-enriched membranes. Freeze-fracture electron microscopy showed that the lipid phase transitions were accompanied by particle aggregation only in the case of palmitic acid-enriched membranes. When the branched-chain acid-enriched membranes were quenched from temperatures below the onset of the lipid phase transition, a random distribution of particles on both fracture faces of the membrane was observed. The membranes were incubated with pig pancreatic phospholipase A₂ at various temperatures. Below the onset of the lipid phase transition phosphatidylglycerol was not accessible for this enzyme in palmitate-enriched membranes. However, a fast hydrolysis of 60–75% of the phosphatidylglycerol could be measured in the branched-chain acid-enriched membranes at temperatures below the onset of the lipid phase transition. The residual phosphatidylglycerol could be hydrolyzed at a slower, temperature-dependent rate. The observations show that lipids containing branched-chain acids undergo a cooperative lipid phase transition which does not result in a tight packing of the lipids of the bilayer below the phase transition.     

On the interaction between intrinsic proteins and phosphatidylglycerol in the membrane of Acholeplasma laidlawii.

About 30% of the phosphatidylglycerol in oleic acid-enriched Acholeplasma laidlawii membranes are not hydrolyzed at temperatures below 10 °C by phospholipase A₂ from porcine pancreas. Removal of 53% of the membrane proteins by proteolysis did not reduce the size of this inaccessible phosphatidylglycerol pool. However, modification of the membrane proteins with 2,4,6-trinitrobenzenesulfonic acid or glutaraldehyde did make an additional 70% of this protected pool of phosphatidylglycerol accessible to phospholipase A₂. Complete hydrolysis of phosphatidylglycerol at low incubation temperatures was achieved only after heat treatment of the membranes which resulted in an extensive aggregation of intrinsic membrane proteins as visualized by freeze-etch electron microscopy. Phospholipase A₂ from bee venom was more effective in hydrolyzing phosphatidylglycerol at low temperature than the pancreatic enzyme. These results show that the inaccessibility of phosphatidylglycerol is not due to resealing of isolated membranes, the presence of a crystalline phase in the membrane lipids, or a shielding effect of surface proteins. The protection against hydrolysis may be due to an interaction of phosphatidylglycerol with intrinsic membrane proteins which is stabilized at low temperatures. Increasing the temperature favors the exchange of protein-bound phosphatidylglycerol with other membrane lipids resulting in complete hydrolysis.     

Phosphorus nuclear magnetic resonance of Acholeplasma laidlawii cell membranes and derived liposomes.

1. The 129 MHz 31P-NMR spectrum of Acholeplasma laidlawii membranes is very similar to the spectrum of the derived liposomes and is a typical "solid state" spectrum in which the major contribution to the linewidth is made by the chemical shift anisotropy. From the value of the chemical shift anisotropy an order parameter of 0.15 is estimated for the lipid phosphates in both membranes. 2. The 31P-NMR spectrum of the A. laidlawii membrane is insensitive to pronase digestion of 4-60% of the membrane proteins and subsequent cytochrome C binding. These results indicate that either no strong lipid polar headgroup-protein interactions occur in the membrane or that the lipid-protein "complexes" in the membrane have a fast rotation (Tc shorter than 10(-6)S) along an axis perpendicular to the plane of the membrane. 3. Phospholipase A2 degrades all the phosphatidylglycerol in the membrane. The resulting membrane contains a phosphoglycolipid as the sole phosphorus-containing compound. The 31P-NMR spectrum of these membranes is identical to the spectrum of the native membranes suggesting a similar motion for the phosphate groups in both lipids. 4. Ca2+ binding to liposomes prepared from either the total polar lipids or the total phosphorus-containing lipids isolated from the A. laidlawii membrane does not affect the 21P-NMR spectrum. 5. The 31P-NMR spectrum of the membranes and derived liposomes, however, is sensitive to lipid phase transitions. When the membrane lipids are in the gel state a broadening of the 31P resonance occurs demonstrating that the polar head group motion in a biological membrane is more restricted below the lipid-phase transition temperature.     

Lipid distribution in Acholeplasma laidlawii membrane. A study using the lactoperoxidase-mediated iodination.

The lactoperoxidase-mediated radioiodination has been applied to study the transbilayer distribution of phospho- and glycolipids in Acholeplasma laidlawii membranes. After radioiodination, about 5% of the 125I-iodine was found in membrane lipids. A comparison of the labeling intensities of the various lipid species between iodinated intact cells and isolated membranes revealed that the glycolipids monoglucosyldiglyceride and diglucosyldiglyceride are located almost exclusively in the outer half of the bilayer, whereas the phospholipids phosphatidylglycerol and diphosphatidylglycerol as well as the phosphoglycolipids glycerophosphoryl-diglucosyldiglyceride and glycerophosphoryl-monoglucosyldiglyceride are almost equally distributed in the outer and inner halves of A. laidlawii membranes.     

Effects of growth at different temperatures on the physical state of lipids in native microsomal membranes from Tetrahymena

Fluorescence measurements of the probe 1,6-diphenyl-1,3,5-hexatriene in native Tetrahymena pyriformis microsomal membranes revealed characteristic "break points" in curves of polarization vs. temperature. In the 5--35 degree C range, membranes from cells grown at 39 degrees C exhibited two break points, one at 11.6 +/- 0.6 degrees C and another at 23.1 +/- 1.6 degrees C. Membranes from 15 degrees C grown cells also showed two break points, one at 8.0 +/- 1.7 degrees C and another at 17.7 +/- 1.7 degrees C. Complementary measurements of turbidity (absorbance at 360 nm) vs. temperature revealed break points at approximately the same temperatures as observed with the fluorescent probe, thus strengthening the likelihood that the break points signify the onset or termination of lipid phase separations or some other significant structural alteration of lipids. In general, break points measured in the native membrane samples occurred at slightly lower temperatures than did break points in lipids extracted from comparable membranes. This suggests two possible types of protein--lipid interaction. First, there may be a selective withdrawal of relatively highly saturated phospholipid molecular species from the bulk lipid phase and into protein annulus regions. Alternatively, the configuration of the hydrophobic core of certain key membrane proteins may be such that nonspecific interactions with the lipids stabilize the liquid-crystalline phase.     

Effects of temperature variation and phenethyl alcohol addition on acyl chain order and lipid organization in Escherichia coli derived membrane systems. A 2H- and 31P-NMR study.

Using 2H- and 31P-NMR techniques the effects of temperature variation and phenethyl alcohol addition were investigated on lipid acyl chain order and on the macroscopic lipid organization of membrane systems derived from cells of the Escherichia coli fatty acid auxotrophic strain K1059, which was grown in the presence of [11,11-2H2]oleic acid. Membranes of intact cells showed a gel to liquid-crystalline phase transition in the range of 4-20 degrees C, which was similar to that observed for the total lipid extract and for the dominant lipid species phosphatidylethanolamine (PE). Phosphatidylglycerol (PG) remained in a fluid bilayer throughout the whole temperature range (4-70 degrees C). At 30 degrees C acyl chain order was highest in PE, followed by the total lipid extract, PG, intact cells, and isolated inner membrane vesicles. Acyl chain order in E. coli PE and PG was much higher than in the corresponding dioleoylphospholipids. E. coli PE was found to maintain a bilayer organization up to about 60 degrees C, whereas in the total lipid extract as well as in intact E. coli cells bilayer destabilization occurred already at about 42 degrees C. It is proposed that the regulation of temperature at which the bilayer-to-non-bilayer transition occurs may be important for membrane functioning in E. coli. Addition of phenethyl alcohol did not affect the macroscopic lipid organization in E. coli cells or in the total lipid extract, but caused a large reduction in chain order of about 70% at 1 mol% of the alcohol in both membrane systems. It is concluded that while both increasing temperature and addition of phenethyl alcohol can affect membrane integrity, in the former case this is due to the induction of non-bilayer lipid structures, whereas in the latter case this is caused by an increase in membrane fluidity.     

Localization of phosphatidylcholine in outer envelope membrane of spinach chloroplasts

We have examined the effects of phospholipase C from Bacillus cereus on the extent of phospholipid hydrolysis in envelope membrane vesicles and in intact chloroplasts. When isolated envelope vesicles were incubated in presence of phospholipase C, phosphatidylcholine and phosphatidylglycerol, but not phosphatidylinositol, were totally converted into diacylglycerol if they were available to the enzyme (i.e., when the vesicles were sonicated in presence of phospholipase C). These experiments demonstrate that phospholipase C can be used to probe the availability of phosphatidylcholine and phosphatidylglycerol in the cytosolic leaflet of the outer envelope membrane from spinach chloroplasts. When isolated, purified, intact chloroplasts were incubated with low amounts of phospholipase C (0.3 U/mg chlorophyll) under very mild conditions (12 degrees C for 1 min), greater than 80% of phosphatidylcholine molecules and almost none of phosphatidylglycerol molecules were hydrolyzed. Since we have also demonstrated, by using several different methods (phase-contrast and electron microscopy, immunochemical and electrophoretic analyses) that isolated spinach chloroplasts, and especially their outer envelope membrane, remained intact after mild treatment with phospholipase C, we can conclude that there is a marked asymmetric distribution of phospholipids across the outer envelope membrane of spinach chloroplasts. Phosphatidylcholine, the major polar lipid of the outer envelope membrane, is almost entirely accessible from the cytosolic side of the membrane and therefore is probably localized in the outer leaflet of the outer envelope bilayer. On the contrary, phosphatidylglycerol, the major polar lipid in the inner envelope membrane and the thylakoids, is probably not accessible to phospholipase C from the cytosol and therefore is probably localized mostly in the inner leaflet of the outer envelope membrane and in the other chloroplast membranes.     

The biosynthetic incorporation of short-chain linear saturated fatty acids by Acholeplasma laidlawii B may suppress cell growth by perturbing membrane lipid polar headgroup distribution

Acholeplasma laidlawii B cells made fatty acid auxotrophic by growth in the presence of the biotin-binding agent avidin grow increasingly poorly at 37 degrees C when supplemented with single exogenous linear saturated fatty acids of decreasing hydrocarbon chain length. Interestingly, this progressive decrease in growth yields with decreasing hydrocarbon chain length is not observed when cells are cultured in the presence of other classes of exogenous fatty acids. Moreover, normal growth is observed is other types of fatty acids with equivalent or shorter hydrocarbon chain lengths, indicating that poor growth in the presence of short-chain linear saturated fatty acids cannot be due to a decrease in membrane lipid bilayer thickness per se. To understand the molecular basis of such growth inhibition, we determined the growth yields, membrane lipid fatty acid and polar headgroups compositions, and phase state and fluidity of the membrane lipids in cells progressively biosynthetically enriched in tridecanoic acid (13:0) or dodecanoic acid (12:0). The growth of fatty acid auxotrophic A. laidlawii B cells grown in the presence of binary combinations of an exogenous fatty acid which supports normal growth on its own and 13:0 or 12:0 revealed that growth inhibition is not observed until 13:0 and 12:0 biosynthetic incorporation levels reach about 90 and 60 mol %, respectively, after which growth is markedly inhibited. Differential scanning calorimetric analyses of membranes from cells maximally enriched in 13:0 indicate that the lipid gel/liquid-crystalline phase transition temperature is unexpectedly high but that at the growth temperature of 37 degrees C, the membrane lipid bilayer is almost exclusively in the liquid-crystalline state but is certainly not excessively fluid. However, high levels of 13:0 incorporation produce a greatly elevated level of the high melting, reversed nonlamellar phase-preferring lipid component monoglucosyl diacylglycerol, and greatly reduced levels of all other membrane lipid components. This marked elevation of monoglucosyl diacylglycerol levels can be rationalized as a regulatory response which maintains the lamellar/nonlamellar phase-forming propensity of the total membrane lipid mixture relatively constant in the face of the biosynthetic incorporation of increasing quantities of short-chain saturated fatty acids, which favor the lamellar phase. However, this lipid biosynthetic response produces a marked decline in the levels of anionic phospholipid and phosphoglycolipid which are probably required to maintain the minimal negative surface charge density of the lipid bilayer, which we suggest is responsible for the observed growth inhibition. This work shows that the lipid biosynthetic regulatory mechanisms present in this organism may sometimes operate at cross purposes such that it is not possible to simultaneously optimize all of the biologically relevant physical properties of the membrane lipid bilayer.     

Hydrolytic action of phospholipases on bacterial membranes.

Substrate specificities of phospholipases C[EC 3.1.4.3] from Clostridium novyi, Clostridium perfringens, Bacillus cereus, and Pseudomonas aureofaciens were studied under the same conditions. Phospholipases C from Clostridium novyi and Bacillus cereus show wide substrate specificities while those of Clostridium perfringens and Pseudomonas aureofaciens show relatively narrow specificities. On the basis of these results, the hydrolytic actions of these phospholipases on membrane lipids of Escherichia coli, Bacillus cereus, and Clostridium novyi were examined under the same conditions. The enzymes of Clostridium novyi and Bacillus cereus attacked all the membranes and their lipid extracts, hydrolyzing phosphatidylethanolamine, phosphatidylglycerol, lyso-phosphatidylethanolamine, and o-aminoacylphosphatidylglycerol. Phospholipase C from Pseudomonas aureofaciens attacked these three membranes and their lipid extracts, hydrolyzing phosphatidylethanolamine. Phospholipase C from Clostridium perfringens hardly attacked the phospholipids of these bacterial membranes. However, phospholipase C from Clostridium perfringens hydrolyzed phosphatidylethanolamine in a mixture containing lipid extract from Escherichia coli membrane and purified phosphatidylcholine from egg yolk.     

Two cholesterol pools in Acholeplasma laidlawii membranes

Cholesterol exchange kinetics between [14C]cholesterol-labeled Acholeplasma laidlawii and Mycoplasma gallisepticum cells and phosphatidylcholine-cholesterol vesicles followed a biphasic curve, with faster exchange rates for A. laidlawii. The same biphasic curve was obtained with isolated membranes. Cholesterol exchange between lipid vesicles and A. laidlawii cells depleted of phospholipids by phospholipase A2, fitted a monophasic linear curve. The data support the hypothesis that the biphasic cholesterol exchange kinetics do not result from the transbilayer distribution of cholesterol, but reflect the presence in the membrane of two cholesterol pools associated with lipids of high and low affinity for cholesterol.     

Characterization of sodium transport in Acholeplasma laidlawii B cells and in lipid vesicles containing purified A. laidlawii (Na+-Mg2+)-ATPase by using nuclear magnetic resonance spectroscopy and 22Na tracer techniques.

The active transport of sodium ions in live Acholeplasma laidlawii B cells and in lipid vesicles containing the (Na+-Mg2+)-ATPase from the plasma membrane of this microorganism was studied by 23Na nuclear magnetic resonance spectroscopic and 22Na tracer techniques, respectively. In live A. laidlawii B cells, the transport of sodium was an active process in which metabolic energy was harnessed for the extrusion of sodium ions against a concentration gradient. The process was inhibited by low temperatures and by the formation of gel state lipid in the plasma membrane of this organism. In reconstituted proteoliposomes containing the purified (Na+-Mg2+)-ATPase, the hydrolysis of ATP was accompanied by the transport of sodium ions into the lipid vesicles, and the transport process was impaired by reagents known to inhibit ATPase activity. At the normal growth temperature (37 degrees C), this transport process required a maximum of 1 mol of ATP per mol of sodium ion transported. Together, these results provide direct experimental evidence that the (Na+-Mg2+)-ATPase of the Acholeplasma laidlawii B membrane is the cation pump which maintains the low levels of intracellular sodium characteristic of this microorganism.     

Action of Phospholipase A2 and Phospholipase C on Bacillus subtilis Protoplasts

Protoplasts prepared from Bacillus subtilis by lysozyme digestion lysed in the presence of pure pancreatic phospholipase A(2). The phospholipids cardiolipin, phosphatidylethanolamine, phosphatidylglycerol and lysylphosphatidylglycerol, which are present in the membrane, are degraded by phospholipase A(2) only after removal of the cell wall, giving free fatty acids and lyso derivatives. The four phospholipids are hydrolyzed equally well at a given enzyme concentration. Differences in the phospholipid composition of the protoplasts were obtained by variations in the growth medium, time of harvesting, and preincubation time with lysozyme. The extent of hydrolysis appeared to depend on the initial phospholipid composition. A relative increase in acidic phospholipids in the membrane facilitated the action of phospholipase A(2), whereas the rate of hydrolysis was diminished when protoplasts were tested which contained a relatively high amount of positively charged phospholipid. Pure phospholipase C from B. cereus preferentially hydrolyzed phosphatidyl-ethanolamine in the B. subtilis membrane. More than 80% of this phospholipid was converted into diglyceride, whereas only 30% of the cardiolipin was hydrolyzed. Such a loss of phospholipids, however, was not followed by lysis of the protoplasts. Liposomes were prepared from the lipid extracts of B. subtilis and incubated with both phospholipases. The hydrolysis pattern of the phospholipids in these model membrane systems was identical to the hydrolysis pattern of the phospholipids in the protoplast membrane. Phospholipase A(2) hydrolyzed all the phospholipids in the liposomes equally well, whereas phospholipase C preferentially degraded phosphatidylethanolamine.     

Effect of cholesterol and lanosterol on the structure and dynamics of the cell membrane of Mycoplasma capricolum. Deuterium nuclear magnetic resonance study.

Deuterium nuclear magnetic resonance (NMR) techniques were employed to study the effect of sterols on the composition and dynamics of the membrane lipids of Mycoplasma capricolum, a natural fatty acid auxotroph that requires sterols for growth. The membrane lipids of cells grown in modified Edwards medium supplemented with cholesterol, oleic acid (OA), and palmitic acid (PA) were composed primarily of phosphatidylglycerol (PG) (60%) and cardiolipin (CL) (35%). The incorporation of cholesterol and the cellular OA/PA ratio increased nonlinearly with increases in exogenous cholesterol level, whereas the levels of phospholipid increased only slightly. At the growth temperature, 37 degrees C, the residual deuterium quadrupole splittings were found to be 43-46 kHz for cells grown with (7,7,8,8-2H4) PA and 1.25 micrograms/ml (30 mol%) to 10 micrograms/ml (50 mol%) cholesterol, respectively, similar to that found in the cholesterol/lecithin binary dispersions of similar cholesterol contents. Deuterium T2e of these samples were found to be 170 +/- 10 microseconds and were independent of cellular cholesterol content. In comparison, T2e of the corresponding lipid extracts were longer (320-420 microseconds) and dependent on cholesterol content. Thus, lipid-protein interactions in the cell membrane is the dominant mechanism responsible for the reduced T2e. At lower temperatures, spectra indicative of the coexistence of gel and liquid-crystalline states were observed for cells having low cholesterol levels. For both cell membrane and membrane lipid extract containing 50 mol% cholesterol, T2e was found to be constant at the temperature range from 15 to 40 degrees C. On the other hand, T2e of cell membrane containing 30 mol% cholesterol decreased linearly at 3.2 microseconds/degrees C. T2e of the corresponding lipid extract showed much stronger temperature variation. Cells containing 39 mol% lanosterol were found to have a quadrupole splitting of 39 kHz, broader than that of the cholesterol-free lecithin dispersion (less than 30 kHz) but less than that of cell membrane containing 30 mol% cholesterol (43 kHz). T2e of the lanosterol sample was found to be 130 +/- 10 microseconds which decreased linearly at a slope similar to that observed for the low cholesterol sample. Therefore, although lanosterol appeared to be capable of modulating cell membrane physical properties it is less effective than cholesterol. When growth rates were correlated with NMR parameters, we found that the membranes of faster growing cells were also more ordered. In contrast, the T2e of the cells of M. capricolum seemed to be maintained at a relatively constant value around 170 microseconds.     

Isolation of purified brush-border membranes from rat jejunum containing a Ca2+-independent phospholipase A2 activity

A novel phospholipase activity was recognized in intact, rat jejunal brush-border membranes and its effect on membrane lipid composition was evaluated following various incubation protocols. Brush-border membranes were isolated from mucosal scrapings by a combination of existing techniques. A brush-border plus nuclei fraction was first prepared by homogenization and low-speed centrifugation in isotonic mannitol, in the presence of 5 mM EDTA. Brush-border membrane vesicles were isolated from this fraction by homogenization, followed by precipitation of the remaining undesired membranes with 10 mM CaCl2. Membranes were judged to be highly purified by marker enzyme content, protein profile, and electron microscopy. In total lipid extracts, prepared immediately following membrane isolation, the ethanolamine phosphatides were found to be the major phospholipid class, accounting for nearly 45% of the total lipid phosphorus. Storage of the intact membranes, at either room temperature or at -20 degrees C, but not at -70 degrees C, resulted in a gradual and progressive hydrolysis of phosphatidylethanolamine to lysophosphatidylethanolamine. Over 60% of the total ethanolamine phospholipid was converted to the lyso form during a 2 week storage period. Incubation of the intact membranes at 37 degrees C produced a similar effect in one hour. Only small amounts of other glycerophospholipids were degraded under these conditions. Hydrolysis was specific for the sn-2 position as more than 80% of the fatty acids in the lysophosphatidylethanolamine were found to be saturated. Substitution of MgCl2 for CaCl2 in the precipitation step did not block the hydrolysis. It was concluded that rat brush-border membranes contain a Ca2+-independent phospholipase A2 with a high substrate preference for phosphatidylethanolamine. The physiological significance of this enzyme is not known.     

Factors affecting steroid and prostaglandin secretion by reproductive tissues of cycling and pregnant sows in vitro

Mitochondrial, microsomal and pellicular membranes were isolated from Tetrahymena cells grown at 39 degrees C or 15 degrees C, and phospholipids, in turn, were separated from total lipids extracted from these membranes. The effect of growth temperature on their solid-to-fluid phase transition temperature was examined by wide-angle X-ray diffraction. The transition temperatures of phospholipids from mitochondria, microsomes and pellicles were 21, 19 and 26 degrees C for cells grown at 39 degrees C and -8, -3 and 6 degrees C for cells grown at 15 degrees C, respectively. All phospholipids were found in a completely fluid state at these growth temperatures. From a comparison between the phospholipids and total lipids from pellicles of cells grown at 39 degrees C, a triterpenoid alcohol, tetrahymanol, caused the transition temperature to increase. The alignment of tetrahymanol in membranes was examined with pellicle'a total lipid oriented in a sample holder.     

Detection of a phosphatidylinositol-specific phospholipase C at the surface of Swiss 3T3 cells and its potential role in the regulation of cell growth

The metabolism and intracellular distribution of a fluorescent analog of phosphatidylinositol (PI), 1,2-[oleoyl,N-(6-[(7-nitrobenz-2-oxa-1,3-diazo-4-yl) aminocaproyl)]-PI (C6-NBD-PI), was examined in monolayer cultures of Swiss 3T3 cells following its insertion into the plasma membrane. Evidence is presented that the exogenously supplied C6-NBD-PI was hydrolyzed by a calcium-dependent PI-specific phospholipase C (PI-PLC) at the external cell surface and that this PI-specific phospholipase C may play a role in the density-dependent inhibition of cell growth: (i) When confluent monolayer cultures were incubated with C6-NBD-PI for 60 min at 7 degrees C, the lipid spontaneously transferred to the cells, and prominent labeling of intracellular membranes was observed. Lipid extraction and analysis demonstrated that more than 60% of the fluorescent lipid in these cells was fluorescent diacylglycerol (DAG). However, when the corresponding fluorescent analogs of phosphatidylcholine or phosphatidylethanolamine were used, the fluorescent lipids readily transferred to cells, but no hydrolysis to fluorescent DAG occurred. (ii) Both intracellular labeling and hydrolysis of C6-NBD-PI to -DAG were inhibited in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. (iii) When myo-[2-3H]inositol-labeled C6-NBD-PI was incubated with cells, [3H]inositol phosphate was released into the incubation medium, but no water-soluble 3H-labeled products were found associated with the cells. (iv) The level of C6-NBD-PI hydrolysis increased dramatically with increasing density of 3T3 cells in monolayer culture.     

Lateral organization in Acholeplasma laidlawii lipid bilayer models containing endogenous pyrene probes.

In membranes of the small prokaryote Acholeplasma laidlawii bilayer- and nonbilayer-prone glycolipids are major species, similar to chloroplast membranes. Enzymes of the glucolipid pathway keep certain important packing properties of the bilayer in vivo, visualized especially as a monolayer curvature stress ('spontaneous curvature'). Two key enzymes depend in a cooperative fashion on substantial amounts of the endogenous anionic lipid phosphatidylglycerol (PG) for activity. The lateral organization of five unsaturated A. laidlawii lipids was analyzed in liposome model bilayers with the use of endogenously produced pyrene-lipid probes, and extensive experimental designs. Of all lipids analyzed, PG especially promoted interactions with the precursor diacylglycerol (DAG), as revealed from pyrene excimer ratio (Ie/Im) responses. Significant interactions were also recorded within the major nonbilayer-prone monoglucosylDAG (MGlcDAG) lipids. The anionic precursor phosphatidic acid (PA) was without effects. Hence, a heterogeneous lateral lipid organization was present in these liquid-crystalline bilayers. The MGlcDAG synthase when binding at the PG bilayer interface, decreased acyl chain ordering (increase of membrane free volume) according to a bis-pyrene-lipid probe, but the enzyme did not influence the bulk lateral lipid organization as recorded from DAG or PG probes. It is concluded that the concentration of the substrate DAG by PG is beneficial for the MGlcDAG synthase, but that binding in a proper orientation/conformation seems most important for activity.     

Calorimetric and spectroscopic studies of lipid thermotropic phase behavior in liver inner mitochondrial membranes from a mammalian hibernator

Arrhenius plots of various enzyme and transport systems associated with the liver mitochondrial inner membranes of ground squirrels exhibit changes in slope at temperatures of 20-25 degrees C in nonhibernating but not in hibernating animals. It has been proposed that the Arrhenius breaks observed in nonhibernating animals are the result of a gel to liquid-crystalline phase transition of the mitochondrial membrane lipids, which also occurs at 20-25 degrees C, and that the absence of such breaks in hibernating animals is due to a major depression of this lipid phase transition to temperatures below 4 degrees C. In order to test this hypothesis, we have examined the thermotropic phase behavior of liver inner mitochondrial membranes from hibernating and nonhibernating Richardson's ground squirrels, Spermophilus richardsonii, by differential scanning calorimetry and by 19F nuclear magnetic resonance and fluorescence polarization spectroscopy. Each of these techniques indicates that no lipid phase transition occurs in the membranes of either hibernating or nonhibernating ground squirrels within the physiological temperature range of this animal (4-37 degrees C). Moreover, differential scanning calorimetric measurements indicate that only a small depression of the lipid gel to liquid-crystalline phase transition, which is centered at about -5 degrees C in nonhibernating animals and at about -9 degrees C in hibernators, occurs. We thus conclude that the Arrhenius plot breaks observed in some membrane-associated enzymatic and transport activities of nonhibernating animals are not the result of a lipid phase transition and that a major shift in the gel to liquid-crystalline lipid phase transition temperature is not responsible for seasonal changes in the thermal behavior of these inner mitochondrial membrane proteins.