Interaction of short chain and long chain fatty acid phosphoglycerides and bile salts with prothrombin

An equimolar mixture of phosphatidylserine and (dioleoyl)phosphatidylethanolamine could substitute for brain cephalin preparations in the single stage prothrombin assay. However, no clot promoting activity was observed on the addition of any of the individual long chain fatty acid-containing phospholipids. Short chain fatty acid-containing phospholipids, such as diheptanoylphosphatidylcholine, diheptanoylphosphatidylethanolamine, diheptanoylphosphatidic acid, and dihexanoylphosphatidylcholine, or dihexanoylphosphatidylethanolamine were inhibitory under all conditions studied. Similar effects of these two general classes of phospholipids were observed in a two-stage thrombin generation system, in which a mixture of bovine Factor Xa, Factor Va, and Ca²⁺ were interacted with prothrombin.In the presence of 25 mM Ca²⁺, dioleoylphosphatidic acid or brain phosphatidylserine alone, and with other long chain phospholipids, formed complexes with bovine plasma prothrombin. On the other hand, dioleoyl-, diheptanoyl- or dihexanoylphosphatidylcholine under comparable conditions showed no binding to prothrombin. There appeared to be a small degree of binding of diheptanoylphosphatidic acid to prothrombin, but it was insufficient to cause any significant change in apparent molecular weight of prothrombin. A mixture of prothrombin, Factor V, diheptanoylphosphatidic acid/diheptanoylphosphatidylcholine and Ca²⁺ eluted in the void volume of Sephadex G-200, but showed a much reduced coagulant activity. Though a net negative charge on the phospholipid surface is required for phospholipid-protein interactions, this does not necessarily promote coagulant activity.Bile acids and bile salts, such as cholic acid, deoxycholic acid, taurocholic acid, glycocholic acid, lithocholic acid and dehydrocholic acid, exerted varying levels of stimulation on the prothrombin assay and thrombin generation system, but were not as effective as the phospholipids. Interestingly, no interaction of these bile acids or salts with prothrombin was noted in the presence of Ca²⁺. The results of these experiments suggest that negatively charged micelles per se are not sufficient for binding alone and that other chemical and physical characteristics of phospholipids are of prime importance.     

Charge transfer chromatography used to study the interaction between synthetic phospholipids and nonylphenyl-nonylglycolate

The interaction between 8 synthetic phospholipids and the nonionic tenzide nonylphenyl-nonylglycolate was studied by charge transfer chromatography. The method has been improved by carrying out the determination at different organic phase concentrations of eluent and by extrapolating the interactive strength back to pure water. It was suggested that two types of interaction of commensurable strength may exist between the tenzide and the phospholipids; hydrophil-hydrophil interactions between the polar head groups of phospholipids and the hydrophilic ethylene-oxide chain of tenzide; hydrophob-hydrophob interactions between the fatty acid chains of phospholipids and the alkyl chain of tenzide. This last effect strongly depends on the length of the fatty acid chain, but it is independent of the presence of double bonds in the lipophilic region of phospholipid.     

Solubilization of multilamellar liposomes of egg yolk lecithin by the bile salt sodiumtaurodeoxycholate and the effect of cholesterol--a rapid-ultrafiltration study

The solubilization of multilamellar egg yolk lecithin liposomes by sodiumtaurodeoxycholate in aqueous phase was studied by ultrafiltration as a function of time, bile salt and cholesterol concentration. The corresponding equilibrium states were analysed. Complete solubilization was achieved at total bile salt/lecithin molar mixing ratios of approximately 5. The minimum ratio to start solubilization was 0.1, corresponding to a free bile salt concentration of only 5% of the critical micelle concentration (CMC). Mean equilibrium constants for the partition of bile salts between non-filterable aggregates and filterable mixed micelles and also the free bile salt concentration were determined. Sodiumtaurodeoxycholate had a higher affinity for small mixed micelles than for lamellar mixed aggregates especially in the presence of cholesterol, which reduces the degree and rate of the solubilization process. A non-homogeneous distribution of bile salts in the lipid phase was detected at low bile salt concentrations.     

Influence of Phospholipids Structure on the Physicochemical Properties and In Vitro Digestibility of Lactoferrin-Loaded Liposomes

The instability of liposomal delivery system during passaging through the gastrointestinal tract (GIT) stimulates a demand to find a stable liposome. This research studied the implications of different types of phospholipids (different fatty acid chain length and saturation, various head group) on liposomal physiochemical properties and stability in the human GIT. The micropolarity of liposomal membrane increased with the decrease of chain lengths of phospholipids, while the morphology observation revealed that the liposomes formed by different phospholipids showed similar in appearance and shapes. The liposomes formed by C_20:0 deformed more severely in simulated gastric fluid, while others exhibited slight changes in the membrane structure. In simulated intestinal fluid, pancreatic lipase and phospholipase A₂, synergized with bile salts, damaged the bilayers structure of all liposomes, with the entrapped lactoferrin release and hydrolysis. Although the various phospholipid structures lead to some difference on the physicochemical properties (size and micropolarity), the enzymic influence displayed more significance during in vitro digestion compared to the types of wall materials. Current results could provide valuable information for the development of more stable and reliable food-grade liposomes in the GIT.

     

Membranotropic effect of phosphocreatine and its structural analogs]

The effects of phosphocreatine (PCr) and its analogues (creatine, phosphocreatinine, phosphoarginine and inorganic phosphate) on liposomal and erythrocyte membranes and on the sarcolemmal membrane of cardiomyocytes were studied. The ESR spectrum of the spin-labeled probe, 5-doxyl-stearate, incorporated into the membrane were recorded for analysis of the structural order of the phospholipid bilayer of these membranes. PCr and its analogues had no effect on the structure of the phospholipid bilayer in liposomes; this effect was temperature-independent. However, in erythrocyte and sarcolemmal membranes the rigidity of the membranes was increased by these compounds (except for creatine) at temperatures above 38-40 degrees C. Analysis of these and literary data revealed that cardiac cell membranes may be the site of protective action of PCr on the ischemic myocardium. The lack of effect on liposomes may suggest that the membrane-stabilizing effect of PCr depends on the presence of membrane proteins. The compounds under study may influence the lipid-protein interactions by increasing the rigidity of membrane phospholipids. These membranotropic effects may be due to the interaction of charged molecules of the compounds with polar heads of phospholipids and/or polar groups of proteins in the membrane interphase which, in turn, may influence the packing of hydrophobic fatty acid chains.     

The effect of tyrosine conjugation on the critical micellar concentration of free and glycine-conjugated bile salts

We investigated the effect of conjugation with the aromatic amino acid tyrosine on the critical micellar concentration (CMC) of bile salts. The CMC values were determined by surface tension and by dye solubilization. The surface tension measurement employed the Du Nouy ring detachment method and the dye solubilization measurement utilized a water-insoluble dye, 1-O-tolylazo-2-naphthol. We compared the CMC values of the sodium salts of cholyltyrosine (cholylTyr), deoxycholyltyrosine (deoxycholylTyr), deoxycholylglycyltyrosine (deoxycholylGlyTyr) chenodeoxycholyltyrosine (chenodeoxycholylTyr), chenodeoxycholylglycyltyrosine (chenodeoxycholylGlyTyr), cholyldiglycyltyrosine (cholylGlyGlyTyr) and cholylglycyltyrosine (cholylGlyTyr) with their respective glycine conjugated bile salts. Both techniques of CMC determination indicated that tyrosine conjugation to free and glycine-conjugated bile salts reduced their CMC significantly.     

Biliary lipid secretion in the rat during infusion of increasing doses of unconjugated bile acids

The aim of the present study was to examine the secretion of biliary components in rats during infusion of increasing doses of either deoxycholic acid, chenodeoxycholic acid or cholic acid and to test the hypothesis that biliary phospholipids may regulate the hepatic bile acid secretory capacity. Analysis of bile samples, collected every 10 min throughout the infusion period showed that there was an elevation of bile acid, phospholipid, cholesterol and alkaline-phosphodiesterase secretion, with all the bile acids, peaking and then gradually declining. Their secretory rates maximum differed and were inversely related to their detergent strength. However, the secretory rates maximum and total output of phospholipids and cholesterol were similar for all bile acids infused. The per cent contribution of phosphatidylcholine to total bile acid-dependent phospholipid secretion was reduced from 84% (in the pre-infusion period) to 59, 46 and 13% at the end of the cholic acid, chenodeoxycholic acid and deoxycholic acid infusions, respectively. This decrease in the per cent contribution of phosphatidylcholine was associated with an increase in the contribution of both sphingomyelin and phosphatidylethanolamine. The biliary phospholipid fatty acid pattern corroborated these changes in the phospholipid classes. Since sphingomyelin and phosphatidylethanolamine are major phospholipids in bile canalicular and other hepatocellular membranes, the marked increase in their secretion in bile during the infusion of high doses of bile acids may indicate solubilization of membrane phospholipids, resulting in membrane structural changes responsible for the reduced excretory function of the liver.     

In vitro effect of free bile acids on the bile canalicular membrane phospholipids in the rat.

Liver cell plasma membranes of male rats were isolated and separated into two fractions, one rich in bile canalicular membranes (BCM) and the other comprising the rest of the plasma membrane (PM). Aliquots of BCM, PM, and microsomes were incubated with deoxycholic, chenodeoxycholic, or cholic acid at bile acid - membrane phospholipid mole ratios up to 100, and the phospholipid solubilization from the PM and from microsomes was linear and apparently nonselective, while that from BCM was biphasic and distinctly selective. Phosphatidyl choline and phosphatidyl ethanolamine made up 90% of the phospholipids solubilized from the BCM at a bile acid - membrane phospholipid mole ratio sufficient to solubilize about 50% of the total phospholipids of the BCM. Of particular interest was the observation that the molecular species and fatty acid composition of the phospholipids solubilized from the BCM under these experimental conditions were similar to those of bile obtained from the same animal, and were quite unlike those solubilized at higher bile acid - phospholipids mole ratios. The data are discussed in terms of the mechanism of the biliary secretion of phospholipids.     

Molecular dynamics simulations of mixed micelles modeling human bile

Extensive molecular dynamics (MD) simulations of binary systems of phospholipids and bile salts, a model for human bile, have been performed. Recent progress in hardware and software development allows simulation of the spontaneous aggregation of the constituents into small mixed micelles, in agreement with experimental observations. The MD simulations reveal the structure of these micelles at atomic detail. The phospholipids are packed radially with their headgroups at the surface and the hydrophobic tails pointing toward the micellar center. The bile salts act as wedges between the phospholipid headgroups, with their hydrophilic sides exposed to the aqueous environment. The structure of the micelles strongly resembles the previously proposed radial shell model. Simulations including small fractions of cholesterol reveal how cholesterol is solubilized inside these mixed micelles without changing their overall structure.     

Accurate separation of vesicles, micelles and cholesterol crystals in supersaturated model biles by ultracentrifugation, ultrafiltration and dialysis.

Gel filtration with bile salts at intermixed micellar/vesicular concentrations (IMC) in the eluant has been proposed to isolate vesicles and micelles from supersaturated model biles, but the presence of vesicular aggregates makes this method unreliable. We have now validated a new method for isolation of various phases. First, aggregated vesicles and - if present - cholesterol crystals are pelleted by short ultracentrifugation. Cholesterol contained in crystals and vesicular aggregates can be quantitated from the difference of cholesterol contents in the pellets before and after bile salt-induced solubilization of the vesicular aggregates. Micelles are then isolated by ultrafiltration of the supernatant through a highly selective 300 kDa filter and unilamellar vesicles by dialysis against buffer containing bile salts at IMC values. Lipids contained in unilamellar vesicles are also estimated by subtraction of lipid contents in filtered micelles from lipid contents in (unilamellar vesicle+micelle containing) supernatant ('subtraction method'). 'Ultrafiltration-dialysis' and 'subtraction' methods yielded identical lipid solubilization in unilamellar vesicles and identical vesicular cholesterol/phospholipid ratios. In contrast, gel filtration yielded much more lipids in micelles and less in unilamellar vesicles, with much higher vesicular cholesterol/phospholipid ratios. When vesicles obtained by dialysis were analyzed by gel filtration, vesicular cholesterol/phospholipid ratios increased strongly, despite correct IMC values for bile salts in the eluant. Subsequent extraction of column material showed significant amounts of lipids. In conclusion, gel filtration may underestimate vesicular lipids and overestimate vesicular cholesterol/phospholipid ratios, supposedly because of lipids remaining attached to the column. Combined ultracentrifugation-ultrafiltration-dialysis should be considered state-of-the-art methodology for quantification of cholesterol carriers in model biles.     

Characterization of octapeptin-membrane interactions using spin-labeled octapeptin

Octapeptin is a membrane-active peptide antibiotic that contains a C10 fatty acid covalently attached to the peptide through an amide bond. Interactions of octapeptin with bacterial membranes and phospholipids were characterized by using spin-labeling techniques and octapeptin derivatives containing fatty acids of varying chain length. Acyl modification of octapeptin demonstrated that the fatty acid of the antibiotic contributed to the antimicrobial activity of octapeptin and its affinity for membranes. The influence of octapeptin and C2 acyloctapeptin on the rates of ascorbate reduction of several membrane-bound doxyl stearates was also examined. These studies demonstrated that octapeptin increaed the rate of diffusion of ascorbate into the lipid bilayer and suggested that the acyl chain contributed to this activity. In addition, an acyl spin-labeled analogue of octapeptin was prepared and shown to retain biological activity. Spectral analysis showed that octapeptin does not aggregate in solution over a wide concentration range. However, the isotropic splitting constant indicated that the acyl chain of octapeptin is not completely exposed to water. It is proposed that the acyl chain of octapeptin in solution interacts with hydrophobic amino acids in the peptide, which partially shields the acyl chain from water. Spectral features of the spin-labeled antibiotic bound to phospholipid dispersions were consistent with directional binding of octapeptin to lipid bilayers with insertion of the fatty acid into the hydrocarbon domain.     

Solubilization of lipids from hamster bile-canalicular and contiguous membranes and from human erythrocyte membranes by conjugated bile salts.

We have demonstrated in vitro the efficacy of the taurine-conjugated dihydroxy bile salts deoxycholate and chenodeoxycholate in solubilizing both cholesterol and phospholipid from hamster liver bile-canalicular and contiguous membranes and from human erythrocyte membrane. On the other hand, the dihydroxy bile salt ursodeoxycholate and the trihydroxy bile salt cholate solubilize much less lipid. The lipid solubilization by the four bile salts correlated well with their hydrophobicity: glycochenodeoxycolate, which is more hydrophobic than the tauro derivative, also solubilized more lipid. All the dihydroxy bile salts have a threshold concentration above which lipid solubilization increases rapidly; this correlates approximately with the critical micellar concentration. The non-micelle-forming bile salt dehydrocholate solubilized no lipid at all up to 32 mM. All the dihydroxy bile acids are much more efficient at solubilizing phospholipid than cholesterol. Cholate does not show such a pronounced discrimination. Lipid solubilization by chenodeoxycholate was essentially complete within 1 min, whereas that by cholate was linear up to 5 min. Maximal lipid solubilization with chenodeoxycholate occurred at 8-12 mM; solubilization by cholate was linear up to 32 mM. Ursodeoxycholate was the only dihydroxy bile salt which was able to solubilize phospholipid (although not cholesterol) below the critical micellar concentration. This similarity between cholate and ursodeoxycholate may reflect their ability to form a more extensive liquid-crystal system. Membrane specificity was demonstrated only inasmuch as the lower the cholesterol/phospholipid ratio in the membrane, the greater the fractional solubilization of cholesterol by bile salts, i.e. the total amount of cholesterol solubilized depended only on the bile-salt concentration. On the other hand, the total amount of phospholipid solubilized decreased with increasing cholesterol/phospholipid ratio in the membrane.     

Interaction of phospholipids with the detergent-solubilized ADP/ATP carrier protein as studied by spin-label electron spin resonance

The interaction of spin-labeled phospholipids with the detergent-solubilized ADP/ATP carrier protein from the inner mitochondrial membrane has been investigated by electron spin resonance spectroscopy. The equilibrium binding of cardiolipin and phosphatidic acid was studied by titration of the protein with spin-labeled phospholipid analogues using a spectral subtraction protocol for the evaluation of the mobile and immobilized lipid portions. This analysis revealed the immobilization of two molecules of spin-labeled cardiolipin per protein dimer. Phosphatidic acid has a similar affinity for the protein surface as cardiolipin. The lipid-protein interaction was less pronounced with the neutral phospholipids and with phosphatidylglycerol. The importance of the electrostatic contribution to the phospholipid-protein interaction shows up with a strong dependence of the lipid binding on salt concentration. Cleavage by phospholipase A2 and spin reduction by ascorbate of the spin-labeled acidic phospholipids in contact with the protein surface suggest that these lipids are located on the outer perimeter of the protein. At reduced detergent concentration, the protein aggregated upon addition of small amounts of cardiolipin but remained solubilized when more cardiolipin was added. This result is discussed with respect to the aggregation state of the protein in the mitochondrial membrane. It is also tentatively concluded that binding of spin-labeled cardiolipin does not displace the tightly bound cardiolipin of mitochondrial origin, which was detected previously by 31P nuclear magnetic resonance spectroscopy.     

Micellar formation of spin-labeled fatty acyl derivatives of lipophilic muramyl dipeptides and their incorporation into liposomal membranes

A lipophilic muramyl dipeptide (MDP) with a nitroxide moiety in its acyl chain (SL-MDP) and its N-methyl derivative (SL-methyl MDP) were synthesized. The SL-MDPs formed micelles (cmc, 0.1-0.3 mM). The ESR spectra of the SL-MDPs in phosphatidylcholine (PC) liposomes at 25 degrees C consisted of an anisotropic signal and three sharp lines, indicating that both SL-MDPs partitioned between membranes and aqueous phase. The amounts of the SL-MDPs in membranes depended on the phospholipid species and the cholesterol (Chol) content, but no appreciable difference was observed between SL-MDPs. The SL-MDPs partitioned well at 25 degrees C into egg yolk PC liposomes but not into pure dipalmitoylphosphatidylcholine (DPPC), suggesting that the incorporation may be related to the membrane fluidity. Chol enhanced the incorporation into both phospholipids. The mobilities of the SL-MDPs in the membranes were less than that of the corresponding spin-labeled fatty acid. Comparison of the mobilities among SL-MDPs, spin-labeled ganglioside and spin-labeled galactosylceramide showed that the hydrophilicity of the polar group may influence the immobilization of their acyl chains.     

Micelle formation of sodium chenodeoxycholate and solubilization into the micelles: comparison with other unconjugated bile salts

Micellization of sodium chenodeoxycholate (NaCDC) was studied for the critical micelle concentration (CMC), the micelle aggregation number, and the degree of counterion binding to micelle at 288.2, 298.2, 308.2, and 318.2 K. They were compared with those of three other unconjugated bile salts; sodium cholate (NaC), sodium deoxycholate (NaDC), and sodium ursodeoxycholate (NaUDC). The I(1)/I(3) ratio of pyrene fluorescence and the solubility dependence of solution pH were employed to determine the CMC values. As the results, a certain concentration range for the CMC and a stepwise molecular aggregation for micellization were found reasonable. Using a stepwise association model of the bile salt anions, the mean aggregation number (n) of NaCDC micelles was found to increase with the total anion concentration, while the n values decreased with increasing temperature; 9.1, 8.1, 7.4, and 6.3 at 288.2, 298.2, 308.2, and 318.2 K, respectively, at 50 mmol dm(-3). The results from four unconjugated bile salts indicate that the number, location, and orientation of hydroxyl groups in the steroid nucleus are quite important for growth of the micelles. Activity of the counterion (Na(+)) was determined by a sodium ion selective electrode in order to confirm the low counterion binding to micelles. The solubilized amount of cholesterol into the aqueous bile salt solutions increased in the order of NaUDC相似文献   

Hydrophobic interaction of lysophospholipids and bile salts at submicellar concentrations

A series of environment-sensitive, fluorescent-labeled N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-monoacylphosphatidylethano lamine (N-NBD-lysoPE) probes of differing acyl chain length (C12-C18) was used to demonstrate the hydrophobic interaction between lysophospholipids and two different bile salts at concentrations below their respective critical micelle concentrations (cmc's). Formation of submicellar aggregates in the presence of bile salt-phospholipid mixed micelles could facilitate lipid absorption in the intestine. To ensure the use of submicellar lysolipid concentrations in the experiments, the cmc of each fluorescent lysolipid probe was determined by concentration-dependent self-quenching. The cmc values obtained for the various N-NBD-lysoPE probes were as follows (microM): monolauroyl, greater than or equal to 40; monomyristoyl, 4; monopalmitoyl, 0.3; monostearoyl, 0.04. Probe concentrations well below their respective cmc's were used in all experiments. The fluorescence of a solution of each lysolipid probe was monitored as the concentration of bile salt was gradually increased. The increase in fluorescence was taken as a measure of the ability of the bile salt molecules to complex with the probe molecule, thereby increasing the fluorescent yield of the lysolipid probe molecule. Determination of the cmc of the bile salts in the presence of the lysolipid probe was made in parallel with the fluorescence measurement by monitoring the increase in light scattering of the solution. Both bile salts were shown to induce maximal increases in fluorescence of the N-NBD-lysoPE derivatives at concentrations of bile salt well below their respective cmc values, indicating the existence of submicellar lysolipid-bile salt aggregates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cholestasis, metabolism and biliary lipid secretion during perfusion of rat liver with different bile salts.

The biological effects of bile acids depend largely upon their molecular structure. When bile acid uptake exceeds the maximal biliary secretory rate (SRm) cholestasis occurs. In order to characterize the influence of bile acid structure on its cholestatic potency we systematically studied SRm, maximal bile flow, maximal and cumulative phospholipid and cholesterol secretion with different taurine-conjugated tri-, di- and keto bile acids (Table I) in the isolated perfused rat liver. Bile acids with a high critical micellar concentration (CMC) promoted the greatest bile flow; a positive non-linear correlation between CMC and maximal bile flow was found. 3 alpha-Hydroxylated bile acids with a hydroxyl group in 6 alpha and/or 7 beta position and lacking a 12 alpha hydroxy group had a high SRm. SRm was not related to CMC or maximal bile flow, respectively. Phospholipids and cholesterol were secreted in a nearly fixed ratio of 12:1; a strong linear relationship could be observed. Cumulative phospholipid secretion over 48 min was significantly lower for non and poor micelle forming bile acids (TDHC and TUC) than for those with comparatively low CMC values (TUDC, TC, THC, THDC, TCDC) (70-140 vs. 210-450 nmol/g liver). At SRm all bile acids with good micelle forming properties showed a similar cumulative biliary lipid output. However, when biliary lipid output was related to 1 mumol bile acid secreted bile acids with a low SRm induced the highest lipid secretion (TCDC, TC). These data (1) demonstrate that a 6 alpha and/or a 7 beta hydroxy group on the steroid nucleus reduce cholestatic potency if the 12 alpha hydroxy group is absent, (2) suggest that in the case of micelle forming bile acids the total amount of phospholipids secreted in bile (depletion of cellular phospholipids) is associated with the occurrence of cholestasis whereby bile acids with a low SRm deplete the cellular phospholipid content at much lower bile acid concentrations than those with a higher SRm and (3) imply that bile acids with non and poor micelle forming properties (TDHC, TUC) presumably do not cause cholestasis (solely) by depletion of cellular phospholipids.     

The outer membrane of Proteus mirabilis. II. The extractable lipid fraction and electron-paramagnetic resonance analysis of the outer and cytoplasmic membranes.

1. The lipid fraction extracted from the outer and cytoplasmic membranes of Proteus mirabilis with chloroform/methanol consisted almost entirely of phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol. 2. The phospholipid content of the cytoplasmic membrane was more than twice that of the outer membrane (38% as against 18% of the total dry weight) and the proportions of the three phospholipids differed somewhat in the two membranes. Yet, the fatty acid composition of the extractable lipids was essentially the same in both membranes. 3. The freedom of motion of spin-labeled fatty acids in the outer membrane of P. mirabilis depended markedly on temperature and on the position of the nitroxide group on the hydrocarbon chain of the probe, suggesting that the local environment of the probe is an associate lipid structure with the properties of a bilayer. Nevertheless, the mobility of the probe was more restricted in the outer membrane than in the cytoplasmic membrane, indicating a higher viscosity of the outer membrane. 4. Chloroform/methanol completely removed the phospholipids from the outer membrane, leaving the lipopolysaccharide moiety intact. The motion of spin-labeled fatty acids in the extracted membranes was, however, highly restricted, suggesting that, in the native outer membrane, the local environment of the probe is composed of phospholipids rather than lipopolysaccharide. Aqueous acetone extraction removed only 75-80% of the phospholipids of the outer membrane. Nevertheless, the mobility of the spin-labeled fatty acid remained highly restricted, suggesting the existence of two phospholipid environments in the outer membrane differing in the nature of their association with the lipopolysaccharide and protein moieties.     

Phospholipid lamellae are cholesterol carriers in human bile

Cholesterol solubility and precipitation in bile are major factors in the pathogenesis of cholesterol gallstones. At present, mixed micelles and phospholipid vesicles are considered to be the only cholesterol carriers in bile. In this study we present evidence showing that phospholipid lamellae are major cholesterol carriers in human bile. Lamellae are a known aggregational form in pure phospholipid model systems. In the present study, lamellae were demonstrated by electron microscopy after negative staining and by small-angle X-ray diffraction in all human gallbladder bile samples examined. During diffraction experiments, cholesterol was found to crystallize from these lamellae. Cholesterol carriers in bile were separated by high-resolution chromatography and by prolonged ultracentrifugation. Lamellae were shown to solubilize most of the biliary cholesterol; vesicles solubilized a lesser amount; while micelles solubilized only a minor portion. Our data suggest that phospholipid aggregates are the main cholesterol carriers in bile. Bile salts may control the equilibrium between the various aggregational forms of cholesterol-carrying phospholipids.     

Specificity in the interaction of phospholipids and fatty acids with vesicle reconstituted cytochrome P-450. A spin label study

The association of fatty acids, androstane, phosphatidylcholine, phosphatidylethanolamine, and phosphatidic acid with purified and phospholipid-vesicle reconstituted cytochrome $\text{P-450}$ was studied by spin labeling. Spin-labeled fatty acids were found to be motionally restricted by cytochrome $\text{P-450}$ in both phospholipid vesicles and in microsomes to a much greater extent than spin-labeled phospholipids. The equilibrium of spin-labeled fatty acid between the bulk membrane lipid and the protein interface could be shifted towards an increased amount in the bulk phospholipid phase by the addition of oleic acid or lysophosphatidylcholine, but not by sodium cholate. Microsomes from different animals showed a variable extent of motional restriction of fatty acids, independent of pretreatment of the animals with phenobarbital or β-naphthoflavone, of cytochrome $\text{P-450}$ content, of the presence of type I and type II substrates for cytochrome $\text{P-450}$. These differences are attributed to the presence of varying amounts of lipid breakdown products in the microsomal membrane such as lysolipids or fatty acids which compete with the externally added spin-labeled fatty acids, or with spin-labeled androstane for the binding to cytochrome $\text{P-450}$. The negative charge of the fatty acid was found to be involved in its association with the protein. Cytochrome $\text{P-450}$ was shown to interact only with a few spin-labeled phospholipid molecules in such a way that the motional restriction of the spin acyl chains can be detected by electron paramagnetic resonance ($\text{τ}{}_{\mathrm{<mtext>R</mtext>}}\text{> 10}{}^{\mathrm{?8}}\text{s}$). The number of associated lipid molecules per protein probably is too small to form a complete shell around the protein. This lipid-protein interaction could be destroyed by the addition of sodium cholate, in contrast to the fatty acid-protein interaction.