On the solubility of calcium deoxycholate: kinetics of precipitation and the effect of conjugated bile salts and lecithin

In view of the low solubility of calcium deoxycholate and the possible induction of cholesterol precipitation in the gallbladder by calcium insoluble salts, we find it of interest to study the precipitation of calcium deoxycholate and its dependence on other bile components. The findings of these studies were as follows: (i) Precipitation of calcium deoxycholate from mixtures of calcium chloride and monomeric deoxycholate (at concentrations below the critical micelle concentration (CMC] is very slow even at relatively high CaCl2 concentrations (more than 20 days at 50 mM CaCl2). (ii) At higher deoxycholic acid (DOC) concentrations, precipitation of micellar DOC is faster and requires much lower calcium chloride concentrations. For any given calcium concentration, the rate of precipitation is maximal at an optimal DOC concentration. In solutions containing 150 mM NaCl, the maximal rate of precipitation occurs at about 10 mM DOC, almost independent of Ca2+ concentration. At lower ionic strength (10 mM NaCl), the optimal DOC concentration is 30 mM. These observations suggest that the most important factors in determining the rate of Ca(DOC)2 precipitation are (a) the ratio between calcium ions bound to the surface of a DOC micelle, and the [DOC] (the Ca2+/DOC binding ratio) and (b) the concentration of DOC micelles. (iii) In the presence of conjugated deoxycholates, the crystallization of calcium deoxycholate is inhibited. Phosphatidylcholine has a similar, although smaller, inhibitory effect. Upon precipitation of calcium deoxycholate from a mixed micellar system containing sodium deoxycholate, phosphatidylcholine and cholesterol, the latter two components spontaneously form vesicles. The anti-nucleating effect of PC and conjugated bile salts is explained in terms of "poisoning" of the crystallization process. In view of the latter results we conclude that under normal conditions calcium deoxycholate is not likely to precipitate in the gallbladder.     

Biliary secretion of phosphatidylcholine and its molecular species in cholecystectomized T-tube patients: effects of bile acid hydrophilicity

The aim of the present study was to establish whether the oral administration of bile acids with different hydrophilic properties affects the amount of phosphatidylcholine as well as the pattern of PC molecular species secreted in bile. We studied the biliary output of total and individual PC species in cholecystectomized T-tube patients, with a total biliary outflow, after oral administration of 750 mg of ursodeoxycholate (3 patients) or deoxycholate (3 patients). The latter experiments were repeated after 3 days of taurine supplementation (1500 mg daily) in order to increase, by means of the tauro-conjugation, the hydrophilicity of the secreted BA. A linear function was observed, during all the studies, between BA and PC biliary secretion, but the amount of PC secreted per mole of BA was higher for the less hydrophilic BA, such as deoxycholate, than for the more hydrophilic ursodeoxycholate or during deoxycholate plus taurine experiments. With regard to the pattern of PC molecular species, we observed no changes after administration of ursodeoxycholate. An increase in the secretion of the major polyenoic species (i.e., 16:0-18:2 and 16:0-20:4), with respect to the secretion of the monoenoic, was revealed during deoxycholate experiments. Conversely, during the deoxycholate plus taurine experiments, the secretion of the major monoenoic PC species (i.e., 16:0-18:1) increased more than that of the polyenoic species. We suggest that the observed modifications of the pattern of PC molecular species, secreted in bile, represent the result of a physicochemical effect of BA on liver membranes.     

Behavior of cholesterol and spin-labeled cholestane in model bile systems studied by electron spin resonance and synchrotron x-ray.

The behavior of mixed bile salt micelles consisting of sodium taurocholate, egg phosphatidylcholine, and cholesterol has been studied by ESR spin labeling and synchrotron x-ray scattering. Consistent with published phase diagrams, pure and mixed bile salt micelles have a limited capacity to incorporate and, hence, solubilize cholesterol. Excess cholesterol crystallizes out, a process that is readily detected both by ESR spin labeling using 3-doxyl-5 alpha-cholestane as a probe for cholesterol and synchrotron x-ray scattering. Both methods yield entirely consistent results. The crystallization of cholesterol from mixed bile salt micelles is indicated by the appearance of a magnetically dilute powder spectrum that is readily detected by visual inspection of the ESR spectra. Both the absence of Heissenberg spin exchange and the observation of a magnetically dilute powder spectrum provide evidence for the spin label co-crystallizing with cholesterol. In mixed bile salt micelles containing egg phosphatidylcholine, the solubility of cholesterol is increased as detected by both methods. With increasing content of phosphatidylcholine and increasing mole ratio cholesterol/phosphatidylcholine, the anisotropy of motion of the spin probe increases. The spin label 3-doxyl-5 alpha-cholestane is a useful substitute for cholesterol provided that it is used in dilute mixtures with excess cholesterol: the cholesterol/spin label mole ratio in these mixtures should be greater than 100. Despite the structural similarity between the two compounds, there are still significant differences in their physico-chemical properties.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stability of mixed micellar bile models supersaturated with cholesterol.

The maximal equilibrium solubility of cholesterol in mixtures of phosphatidylcholine (PC)1 and bile salts depends on the cholesterol/PC ratio (Rc) and on the effective ratio (Re) between nonmonomeric bile salts and the sum (CT) of PC and cholesterol concentrations (Carey and Small, 1978; Lichtenberg et al., 1984). By contrast, the concentration of bile salts required for solubilization of liposomes made of PC and cholesterol does not depend on Rc (Lichtenberg et al., 1984 and 1988). Thus, for Rc greater than 0.4, solubilization of the PC-cholesterol liposomes yields PC-cholesterol-bile salts mixed micellar systems which are supersaturated with cholesterol. In these metastable systems, the mixed micelles spontaneously undergo partial revesiculation followed by crystallization of cholesterol. The rate of the latter processes depends upon Rc, Re, and CT. For any given Rc and Re, the rate of revesiculation increases dramatically with increasing the lipid concentration CT, reflecting the involvement of many mixed micelles in the formation of each vesicle. The rate also increases, for any given CT and Re, upon increasing the cholesterol to PC ratio, Rc, probably due to the increasing degree of supersaturation. Increasing the cholate to lipid effective ratio, Re, by elevation of cholate concentration at constant Rc and CT has a complex effect on the rate of the revesiculation process. As expected, cholate concentration higher than that required for complete solubilization at equilibrium yields stable mixed micellar systems which do not undergo revesiculation, but for lower cholate concentrations decreasing the degree of supersaturation (by increasing [cholate]) results in faster revesiculation. We interpret these results in terms of the structure of the mixed micelles; micelles with two or more PC molecules per one molecule of cholesterol are relatively stable but increasing the bile salt concentration may cause dissociation of such 1:2 cholesterol:PC complexes, hence reducing the stability of the mixed micellar dispersions. The instability of PC-cholesterol-cholate mixed systems with intermediary range of cholate to lipids ratio may be significant to gallbladder stone formation as: (a) biliary bile contains PC-cholesterol vesicles which may be, at least partially, solubilized by bile salts during the process of bile concentration in the gallbladder, resulting in mixtures similar to our model systems; and (b) the bile composition of cholesterol gallstone patients is within an intermediary range of bile salts to lipids ratio.     

Parameters affecting the accuracy of the MALDI-TOF MS determination of the phosphatidylcholine/lysophosphatidylcholine (PC/LPC) ratio as potential marker of spermatozoa quality

Matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is increasingly used to characterize (phospho)lipids. However, quantitative MALDI data are often questioned because ion suppression may occur if mixtures are analyzed. Therefore, relative (but no absolute) data are normally derived from the MALDI mass spectra of lipid mixtures. We are particularly interested in the phosphatidylcholine/lysophosphatidylcholine (PC/LPC) ratio because it seems to represent a suitable measure of the inflammatory activity. In this study, different parameters affecting the achievable accuracy of the MALDI-TOF MS determination of the PC/LPC ratio are compared. It will be shown that particularly the applied laser fluence as well as the used solvents influence the accuracies. Using artificial lipid mixtures it will be demonstrated that the PC/LPC ratio can be determined with an accuracy of about ±10% making the MALDI assay comparable to established methods. Finally, it will be shown that the optimized conditions are also useful to determine the PC/LPC ratios in human seminal plasma.     

A study of the physicochemical interactions between biliary lipids and chlorpromazine hydrochloride. Bile-salt precipitation as a mechanism of phenothiazine-induced bile secretory failure.

Since chlorpromazine hydrochloride [2-chloro-10-(3-dimethylaminopropyl)-phenothiazine hydrochloride] is commonly implicated in causing bile-secretory failure in man and is secreted into bile, we have studied the physicochemical interactions of the drug with the major components of bile in vitro. Chlorpromazine hydrochloride molecules are amphiphilic by virtue of possessing a polar tertiary amine group linked by a short paraffin chain to a tricyclic hydrophobic part. At pH values below the apparent pK (pK'a 7.4) the molecules are water-soluble cationic detergents. We show that bile salts in concentrations above their critical micellar concentrations are precipitated from solution by chlorpromazine hydrochloride as insoluble 1:1 salt complexes. In the case of mixed bile-salt/phosphatidylcholine micellar solutions, however, the degree of precipitation is inhibited by the phospholipid in proportion to its mole fraction. With increases in the concentration of chlorpromazine hydrochloride or bile salt, micellar solubilization of the precipitated complexes results. Sonicated dispersions of the negatively charged phospholipid phosphatidylserine were also precipitated, but dispersions of the zwitterionic phospholipid phosphatidylcholine were not. Chlorpromazine hydrochloride efficiently solubilized these membrane phospholipids as mixed micellar solutions when the drug:phospholipid molar ratio reached 4:1. Polarizing-microscopy and X-ray-diffraction studies revealed that the precipitated complexes were amorphous and potentiometric studies confirmed the presence of a salt bond. Some dissociation of the complex occurred in the case of the most polar bile salt (Ks 0.365). As canalicular bile-salt secretion determines much of bile-water flow, we propose that complexing and precipitation of bile salts by chlorpromazine hydrochloride and its metabolites may be physicochemically related to the reversible bile-secretory failure produced by this drug.     

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   

Polydispersity of aggregates formed by the polyene antibiotic amphotericin B and deoxycholate. A spin label study

The amphotericin B-deoxycholate (AB-DOC) system (1:2, mole basis) was studied with regard to its organizational properties making use of spin label ESR spectra. The spectra of a fatty acid spin label intercalated in AB-DOC preparations revealed two components, one strongly (S) and one weakly (W) immobilized. Spectral subtractions indicated that S corresponds to label in mixed AB-DOC aggregates while W is due to label in deoxycholate micelles. This situation, coexistence of different aggregates, is similar to that found in systems consisting of bile salts and phospholipids. The DOC/AB mole ratio in the mixed aggregate is highest when pure DOC micelles are present. Dilution leads to disappearance of the latter and to continuous loss of DOC from AB-DOC accompanied by an increase in size and decrease in solubility of the aggregates, as verified by filtration and centrifugation experiments. The results indicate that AB-DOC systems are polydisperse. Since amphotericin B preparations having different organizational properties display different toxic and therapeutic effect, the study of amphotericin B aggregates should help in understanding these phenomena at a molecular level.     

Solubilization of cholesterol and polycyclic aromatic compounds into sodium bile salt micelles (part 2)

The aqueous solubility of cholesterol was determined over the temperature range from 288.2 to 318.2 K with intervals of 5 K by the enzymatic method. The solubility was (3.7+/-0.3)x10(-8) mol dm(-3) (average +/- S.D.) at 308.2 K. The maximum additive concentrations of cholesterol into the aqueous micellar solutions of sodium deoxycholate (NaDC), sodium ursodeoxycholate (NaUDC), and sodium cholate (NaC) were spectrophotometrically determined at different temperatures. The cholesterol solubility increased in the order of NaUDC相似文献   

Cholesterol modulation of lipid intermixing in phospholipid and glycosphingolipid mixtures. Evaluation using fluorescent lipid probes and brominated lipid quenchers.

Carbazole- and indole-labeled phospholipids have been used to monitor the homo- or heterogeneity of lipid mixing in several types of lipid bilayers combining a brominated and a nonbrominated lipid with varying amounts of cholesterol. Experimental quenching curves (relating the normalized probe fluorescence intensity to the mole fraction of brominated lipid) show a characteristic smooth, monophasic form for homogeneous liquid-crystalline lipid mixtures. However, for mixtures exhibiting lipid lateral segregation, such curves show marked perturbations in form over the region of composition where segregation occurs. Using this approach, it is found that high mole fractions of cholesterol (40-50 mol %) promote the formation of apparently homogeneous solutions in mixtures of disaturated and monounsaturated phosphatidylcholines (PCs) that exhibit extensive thermotropic phase separations in the absence of sterol. At only slightly lower levels of cholesterol, however, these systems exhibit inhomogeneous lipid mixing over a wide range of relative proportions of the two PC components. Mixtures of cerebroside and monounsaturated PCs, even at high bilayer cholesterol contents, exhibit significant inhomogeneity in lipid mixing over a wide range of cerebroside/PC ratios. Phase-separating PC/PC and PC/cerebroside mixtures can readily form long-lived metastable solutions when the level of the higher-melting component in the liquid-crystalline phase exceeds its equilibrium solubility by as much as 20-30 mol %; this tendency is significantly increased by cholesterol. Cholesterol shows no significant ability to enhance lipid intermixing in a third type of phase-separating lipid system, combining a monounsaturated PC with a monounsaturated phosphatidic acid--calcium complex. Experiments using cleavable phospholipid conjugates, linking a fluorescent lipid to a brominated lipid, suggest that each fluorescent molecule probes a local lipid domain comprising approximately less than 40-50 nearby acyl chains.     

Binding of bile salts to pancreatic colipase and lipase.

The binding of conjugated bile salts to pancreatic colipase and lipase has been studied by equilibrium dialysis and gel filtration. The results indicate that at physiological ionic strength and pH, conjugated bile salts bind as micelles to colipase: 12-15 moles/mole of colipase for the dihydroxy conjugates and 2-4 for the trihydroxy conjugates. No binding of bile salt takes place from monomeric solutions. Under the same experimental conditions, only 1-2 moles of conjugated dihydroxy bile salts bind to pancreatic lipase.     

Effects of bile salts and prostaglandins on sodium transport in isolated rat gastric mucosa

To determine whether prostaglandins may protect against bile salt inhibition of ion transport in the stomach, gastric mucosal tissue was isolated from the rat and mounted in flux chambers. Transport of Na+ was traced with radioisotopes in the absence of bile salts and then in the presence of conjugated taurocholate or unconjugated deoxycholate at low, intermediate and high mucosal concentrations (1, 5 and 15 mmol/1). At a high (7.40) or low (3.4) mucosal pH, only the unconjugated deoxycholate inhibited active Na+ transport from mucosa to submucosa with respect to untreated controls. Inhibition of Na+ transport was apparent at a low level of deoxycholate, which also inhibited the electrical potential difference. Intermediate and high levels of deoxycholate lowered the tissue resistance. When the tissues were exposed to mucosal prostaglandin E2 or its 16,16-dimethyl analogue before and during acidified taurocholate administration, Na+ transport was not changed significantly but the electrical resistance remained high. Thus, unconjugated bile salt is more potent than conjugated bile salt in inhibiting Na+ transport and breaking the gastric mucosal barrier, and prostaglandins may afford some small protection.     

Distinct effects of three bile salts on cholesterol solubilization by oleate-monoolein-bile salt micelles.

Micellar cholesterol solubilities in bile salt-monoolein-oleic acid systems have been determined. Whatever the bile salt/oleyl compounds ratio, taurochenodeoxycholate solubilizes more cholesterol than taurocholate and much more than tauroursodeoxycholate. At pH 6.7, the cholesterol solubility limit is about the same with either oleate or monoolein. Cholesterol solubility falls in oleate-bile acid mixtures as the pH is raised. The capacity for supersaturation with cholesterol is greater for bile salt-monoolein than for bile salt-oleate micelles. For the latter it decreases as pH increases.     

Structural and kinetic studies on the solubilization of lecithin by sodium deoxycholate.

Mixed dispersions of egg phosphatidylcholine (PC) and the bile salt sodium deoxycholate (DOC) were prepared by various methods, and their turbidities and proton magnetic resonance spectra were studied as a function of time. The spectra of dispersions prepared by dissolving both components in a common organic solvent and replacing the organic solvent by water did not change with time, indicating that the mixed aggregates formed represent "a state of equilibrium". In the 1H NMR spectra of these mixed aggregates, only signals from small mixed micellar structures were narrow enough to be observed. The dependence of the NMR line widths on the molar ratio of DOC to PC (R) is interpreted in terms of a model for the PC--DOC mixed micelles, according to which PC is arranged as a curved bilayer, the curvature of which increases with increasing R. Upon mixing PC with aqueous solutions of DOC, we found that the mixed aggregates formed are slowly reorganized and ultimately reach the same state of equilibrium. This reorganization was found to be a pseudo-first-order process, the rate constant of which depends linearly upon the detergent concentration. This process involves saturation of the outer bilayers of the multilamellar PC by detergent, followed by transformation of these bilayers into mixed micelles. It is concluded that the solubilization occurs through consecutive "peeling off" of lecithin bilayers.     

Bile salts in submicellar concentrations promote bidirectional cholesterol transfer (exchange) as a function of their hydrophobicity

Cholesterol, despite its poor solubility in aqueous solutions, exchanges efficiently between membranes. Movement of cholesterol between different subcellular membranes in the hepatocyte is necessary for assembly of lipoproteins, biliary cholesterol secretion, and bile acid synthesis. Factors which initiate and facilitate transfer of cholesterol between different membranes in the hepatocyte are incompletely understood. It is known that cholesterol secretion into the bile is linked to bile salt secretion. In the present study, we investigated the effects of bile salts of different physicochemical properties at submicellar concentrations (150- 600 microM) on the transfer of [14C]cholesterol from hepatocytes, or crude hepatocellular membranes (donors), to rat high density lipoproteins (acceptor). Bile salts included taurine conjugates of ursodeoxycholic acid (TUDCA), hyodeoxycholic acid (THDCA), cholic acid (TCA), chenodeoxycholic acid (TCDCA), and deoxycholic acid (TDCA). High density lipoprotein (HDL) was separated from hepatocellular membranes and the transfer of [14C]cholesterol from the membranes to HDL was quantitatively determined. In the absence of HDL, [14C]cholesterol remained confined to the membrane fraction. Following addition of HDL, [4-14C]cholesterol in the HDL fraction increased linearly over time. Addition of hydrophilic bile salts (TUDCA and THDCA) increased transfer of [4-14C]cholesterol to HDL only minimally. By contrast, more hydrophobic bile salts stimulated transfer of labeled cholesterol to HDL, and their potency increased in order of increasing hydrophobicity (TCA less than TCDCA less than TDCA). Both for single bile salts and mixtures of bile salts at a total bile salt concentration of 0.30 mM, the rate of cholesterol transfer exhibited a strong linear correlation with a bile salt monomeric hydrophobicity index (r = 0.95; P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Rheological studies on mixed phospholipid sols and their interaction with penicillins I. Continuous shear viscometry

The effects of sonication and ageing of phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) sols have been examined. Ageing was found to significantly alter the nature of the flow curves obtained for sonicated mixtures; only after 48 hr was reproducibility obtained. Rheological studies on aged mixed sols indicated behaviour similar to that previously reported. Addition of urea and guanidine hydrochloride in concentrations up to 8 M indicated hydrophobic interaction between PC and LPC.

Addition of Penicillin G and Ampicillin to mixed PC/LPC sols indicated that Penicillin G interacted to a greater degree than Ampicillin; pronounced Penicillin G interaction was observed in a mixed sol containing 60% PC, 40% LPC, a system where the asymmetry of the aggregates is at a minimum. Ampicillin appeared to interact to a greater degree with mixtures containing more LPC than PC. Differences in hydrophobic interaction were indicated by use of urea and guanidine hydrochloride, which is of interest in the light of present knowledge regarding the in vivo activity of these antibiotics.     

Sodium-deoxycholate-assisted tryptic digestion and identification of proteolytically resistant proteins

Identification of proteolytically resistant proteins with compact molecular structure and/or poor water solubility is a challenge in current proteomic study. In this study, sodium deoxycholate (SDC)-assisted tryptic digestion and identification of proteolytically resistant myoglobin and integral membrane proteins were systematically investigated. When the effect of SDC up to 10% on trypsin activity was investigated, little decrease in the trypsin activity was observed in 1% SDC solution, 2-5% SDC decreased the enzyme activity only by about 13.6%, and even in the presence of 10% SDC trypsin still retained 77.4% of its activity. Matrix-assisted laser desorption ionization time of flight mass spectrometry analysis showed that SDC could be removed from sample solution with acid treatment followed by centrifugation, and the remaining SDC, if any, had little effect on mass spectrometry analysis with regard to the number and signal/noise ratio of ions in the mass spectra. Compared with urea and methanol, two other commonly used additives in addition to SDS in proteomic analysis, SDC improved more efficiently the denaturation, solubilization, and tryptic digestion of proteins, particularly proteolytically resistant myoglobin and integral membrane proteins, thereby enhancing the efficiency of their identification with regard to the number of identified proteins and unique peptides and the sequence coverage of matched proteins.     

Further studies on the bile salt induction of 7 alpha- and 7 beta-hydroxysteroid dehydrogenases in Clostridium absonum

Optimal induction of 7 alpha- and 7 beta-hydroxysteroid dehydrogenase in 100-ml cultures grown to stationary phase was achieved by the addition of metabolizable bile salt inducers: chenodeoxycholate, 7-ketolithocholate or cholate at 2.5-3 h after inoculation. Bile salt addition prior to or after this period markedly reduced the enzyme levels induced. However, when the non-metabolizable inducers deoxycholate and 12-ketolithocholate were similarly added, no significant differences in enzyme levels were observed between addition at 2.5-3 h or at earlier times. The ability of both metabolizable and non-metabolizable bile salts to induce the enzymes fell markedly when additions were made later than approximately 3.5 h. Kinetic studies using 1-l cultures suggest that in a larger culture a somewhat earlier inducer addition period is optimal. When ranked according to the level of enzymes induced the order in decreasing induction power was: chenodeoxycholate, 7-ketolithocholate, deoxycholate, 12-ketolithocholate and cholate. Mixtures of cholate and suboptimal concentrations of deoxycholate induced the culture better than the sum of the two concentrations individually. The end product, ursodeoxycholate, was very effective in blocking the induction by chenodeoxycholate or deoxycholate. Ursocholate (3 alpha, 7 beta, 12 alpha-trihydroxy-5 beta-cholanoate) was less effective. Cultures when grown for 3 h with various bile salts or none, then centrifuged and recultured for a further 3 h in fresh medium containing chenodeoxycholate, all yielded identical enzyme levels within experimental error. We conclude that exposure of the organism to bile salt inducer in the last 3 h of culture was important, while the history of the culture prior to this time was unimportant in the induction process.     

Fluid-phase connectivity and translational diffusion in a eutectic, two-component, two-phase phosphatidylcholine bilayer

In recent work [Vaz, W.L.C., Melo, E.C.C., & Thompson, T.E. (1989) Biophys. J. 56, 869-876] we have shown that translational diffusion studies using fluorescence recovery after photobleaching (FRAP) provide information concerning domain structures and fluid-phase connectivity in lipid bilayers in which solid and fluid phases coexist. In the present paper, translational diffusion of the fluid-phase-soluble, solid-phase-insoluble fluorescent lipid derivative N-(7-nitrobenzoxa-2,3-diazol-4-yl) dilauroyl-phosphatidylethanolamine and the fluid-phase connectivity are examined in lipid bilayers prepared from binary mixtures of 1-docosanoyl-2-dodecanoylphosphatidylcholine (C22:0C12:0PC) and 1,2-diheptadecanoylphosphatidylcholine (di-C17:0PC) by using FRAP. The phosphatidylcholine mixture used provides a eutectic system with a eutectic point at a composition of about 0.4 mole fraction of di-C17:0PC and a temperature of about 37 degrees C [Sisk, R.B., Wang, Z.Q., Lin, H.N., & Huang, C.H. (1990) Biophys. J. 58, 777-783]. Two regions in temperature and composition, respectively below and above 0.4 mole fraction of di-C17:0PC, where fluid and solid phases coexist in the same lipid bilayer, are available for examination of fluid-phase connectivity. In mixtures containing less than 0.4 mole fraction of di-C17:0PC the fluid phase coexists with a mixed interdigitated Lc gel phase composed mostly of C22:0C12:0PC, whereas in mixtures containing greater than 0.4 mole fraction of di-C17:0PC the fluid phase coexists with a P beta' gel phase mostly composed of di-C17:0PC. When the solid phase is a P beta' gel phase, the temperature of fluid-phase connectivity for the mixtures lies close to the fluidus, which means that a small (approximately 20%) mass fraction of solid phase can divide the large bulk of the bilayer that is fluid into nonconnected domains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipid-protein ratios by infrared spectroscopy

A method for measuring the ratio of diacyl phospholipid to protein in lipid-protein mixtures and membranes by infrared spectroscopy is described. Samples made of diacyl phospholipid and proteins mixed in known ratios were analyzed for lipid-protein ratio by the infrared (ir) method. Results had a standard deviation of less than +/- 4% over the lipid-protein molar ratio range of 9:1 to 320:1. Calculations of the ratio of total lipid to protein require that the diacyl phospholipid-to-protein ratio be divided by the mole fraction of diacyl phospholipid in the total lipid. Phospholipid-protein ratios for various sarcoplasmic reticulum membrane preparations (R1-washed, octylglucoside purified, deoxycholate treated) were determined by the ir method and compared to literature values. Also, phospholipid-protein ratios were determined for R1-washed sarcoplasmic reticulum by three chemical analyses using different protein assays and were compared with ratios obtained by the infrared method. The infrared results were closest to those of a chemical method designed specifically for membrane proteins.