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There is now a general consensus that the intestinal absorption of water-insoluble, dietary lipids is protein-mediated, but the assignment of protein(s) to this function is still a matter of debate. To address this issue, we measured beta-carotene and cholesterol absorption in wild-type and SR-BI knockout mice and the uptake of these lipids in vitro using brush border membrane (BBM) vesicles. From the comparison of the in vivo and in vitro results we conclude that both BBM-resident class B scavenger receptors, SR-BI and CD36, can facilitate the absorption of beta-carotene and cholesterol. SR-BI is essential for beta-carotene absorption, at least in mice on a high fat diet. This is due to the fact that the absorption of beta-carotene is restricted to the duodenum and SR-BI is the predominant receptor in the mouse duodenum. In contrast, SR-BI may be involved but is not essential for cholesterol absorption in the small intestine. The question of whether SR-BI contributes to cholesterol absorption in vivo is still unresolved. Transfection of COS-7 cells with SR-BI or CD36 confers on these cells lipid uptake properties closely resembling those of enterocytes and BBM vesicles. Both scavenger receptors facilitate the uptake of dietary lipids such as beta-carotene, free and esterified cholesterol, phospholipids, and fatty acids into COS-7 cells. This lipid uptake is effected from three different lipid donor particles: mixed bile salt micelles, phospholipid small unilamellar vesicles, and trioleoylglycerol emulsions which are all likely to be present in the small intestine. Ezetimibe, a representative of a new class of drugs that inhibit intestinal cholesterol absorption, blocks SR-BI- and CD36-facilitated uptake of cholesterol into COS-7 cells.

The kinetics of long-chain fatty acid (FA) transfer from three different donor systems to unilamellar egg phosphatidylcholine (EPC) vesicles containing the pH-sensitive fluorophore pyranine in the vesicle cavity were determined. The transfer of long-chain FA from three FA donors, FA vesicles, unilamellar EPC vesicles containing FA, and bovine serum albumin-FA complexes to pyranine-containing EPC vesicles is a true first-order process, indicating that the FA transfer proceeds through the aqueous phase and not through collisional contacts between the donor and acceptor. A collisional mechanism would be at least bimolecular, giving rise to second-order kinetics. Evidence from stopped-flow fluorescence spectroscopy using the pyranine assay (as developed by Kamp, F., and Hamilton, J. A. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 11367-11370) shows that the transverse or flip-flop motion of long-chain FA (from 14 to 22 C atoms) is immeasurably fast in both small and large unilamellar EPC vesicles and characterized by half-times t(1/2) < 5 ms. The rate-limiting step of FA transfer from these different donor systems to pyranine-containing EPC vesicles is the dissociation or desorption of the FA molecule from the donor. The desorption of the FA molecule is chain-length-dependent, confirming published data (Zhang et al. (1996) Biochemistry 35, 16055-16060): the first-order rate constant k(1) decreases by a factor of about 10 with elongation of the FA chain by two CH(2) groups. Similar rates of desorption are observed for the transfer of oleic acid from the three donors to pyranine-containing EPC vesicles with rate constants k(1) ranging from 0.4 to 1.3 s(-1). We also show that osmotically stressed, pyranine-containing EPC vesicles can give rise to artifacts. In the presence of a chemical potential gradient across the lipid bilayer of these vesicles, fast kinetic processes are observed with stopped-flow fluorescence spectroscopy which are probably due to electrostatic and/or osmotic effects.ne

The serum lipoprotein high-density lipoprotein (HDL), which is a ligand of scavenger receptors such as scavenger receptor class B type I (SR-BI) and cluster determinant 36 (CD36), can act as a donor particle for intestinal lipid uptake into the brush border membrane (BBM). Both cholesterol and phospholipids are taken up by the plasma membrane of BBM vesicles (BBMV) and Caco-2 cells in a facilitated (protein-mediated) process. The protein-mediated transfer of cholesterol from reconstituted HDL to BBMV depends on the lipid composition of the HDL. In the presence of sphingomyelin, the transfer of cholesterol is slowed by a factor of about 3 probably due to complex formation between cholesterol and the sphingolipid. It is shown that the mechanism of lipid transfer from reconstituted HDL to either BBMV or Caco-2 cells as the acceptor is consistent with selective lipid uptake: the lipid donor docks at the membrane-resident scavenger receptors which mediate the transfer of lipids between donor and acceptor. Selective lipid uptake implies that lipid, but no apoprotein is transferred from the donor to the BBM, thus excluding endocytotic processes. The two BBM models used here clearly indicate that fusion of donor particles with the BBM can be ruled out as a major mechanism contributing to intestinal lipid uptake. Here we demonstrate that CD36, another member of the family of scavenger receptors, is present in rabbit and human BBM vesicles. This receptor mediates the uptake of free cholesterol, but not of esterified cholesterol, the uptake of which is mediated exclusively by SR-BI. More than one scavenger receptor appears to be involved in the uptake of free cholesterol with SR-BI contributing about 25% and CD36 about 35%. There is another yet unidentified protein accounting for the remaining 30 to 40%.

Exchangeable serum apolipoproteins and amphipathic alpha-helical peptides are effective inhibitors of sterol (free and esterified cholesterol) uptake at the small-intestinal brush border membrane. The minimal structural requirement of an inhibitor is an amphipathic alpha-helix of 18 amino acids. The inhibition is competitive, indicating that the inhibitor binds to scavenger receptor class B type I (SR-BI) present in the brush border membrane and responsible for sterol uptake. Binding of apolipoprotein A-I to SR-BI of rabbit brush border membrane is cooperative, characterized by a dissociation constant K(d) = 0.45 microM and a Hill coefficient of n = 2.8. The cooperativity of the interaction is due to binding of the inhibitor molecule to a dimeric or oligomeric form of SR-BI held together by disulfide bridges. Consistent with the competitive nature of the inhibition, the K(d) value agrees within experimental error with the IC(50) value of inhibition and with the inhibition constant K(I). After proteinase K treatment of brush border membrane vesicles, the affinity of the interaction of apolipoprotein A-I expressed as K(d) is reduced by a factor of 20, and the cooperativity is lost. The interaction of proteinase K-treated brush border membrane vesicles with apolipoprotein A-I is nonspecific partitioning of the apolipoprotein into the lipid bilayer of brush border membrane vesicles.

Sterol carrier protein 2, also known as nonspecific lipid transfer protein is a ubiquitous, small, basic protein of 13 kDa found in animals. Its primary structure is highly conserved between different species, and it has been implicated in the intracellular transport of lipids and in a wide range of other in vitro functions related to sterol and fatty acid metabolism. Sterol carrier protein 2 deficiency in mice leads to elevated concentrations of phytanic acid in the serum and causes hepatocarcinogenesis. However, its actual physiological role is still unknown. Although sterol carrier protein 2 has been studied extensively in the past 20 years, very little is known concerning its three-dimensional structure. The crystal structure of rabbit sterol carrier protein 2, determined at 1.8 A resolution with the MIRAS method, shows a unique alpha/beta-fold. The core of this protein forms a five-stranded antiparallel beta-sheet flanked by five helices. A C-terminal segment (residues 114-123), together with part of the beta-sheet and four alpha-helices, form a hydrophobic tunnel providing the environment for apolar ligands such as fatty acids and fatty acyl-coenzyme As. Structurally well-characterized nonspecific lipid transfer proteins from plants have hydrophobic tunnel-like cavities, which were identified as the binding site for fatty acids and related apolar ligands. Despite the fact that plant nonspecific lipid transfer proteins are smaller proteins than sterol carrier protein 2, show no sequence homology to sterol carrier protein 2, and are structurally unrelated, the cavities of these two classes of proteins are very similar with respect to size, shape, and hydrophobicity, suggesting a common functional role.

Here we show that scavenger receptor class B type I is present in the small-intestine brush border membrane where it facilitates the uptake of dietary cholesterol from either bile salt micelles or phospholipid vesicles. This receptor can also function as a port for several additional classes of lipids, including cholesteryl esters, triacylglycerols, and phospholipids. It is the first receptor demonstrated to be involved in the absorption of dietary lipids in the intestine. In liver and steroidogenic tissues, the physiological ligand of this receptor is high-density lipoprotein. We show that binding of high-density lipoprotein and apolipoprotein A-I to the brush border membrane-resident receptor inhibits uptake of cholesterol (sterol) into the brush border membrane from lipid donor particles. This finding lends further support to the conclusion that scavenger receptor BI catalyzes intestinal cholesterol uptake. Our findings suggest new therapeutic approaches for limiting the absorption of dietary cholesterol and reducing hypercholesterolemia and the risk of atherosclerosis.

The sterol (free and esterified cholesterol) transport activity of the small-intestinal brush border membrane was solubilized with the short-chain detergent diheptanoylphosphatidylcholine and reconstituted to an artificial membrane system (proteoliposomes). The resulting proteoliposomes were identified as unilamellar membrane vesicles ranging in size between 50 and 200 nm with a broad maximum at 70-110 nm. That the sterol transport protein was indeed incorporated into the lipid bilayer was shown by density gradient centrifugation on a Ficoll gradient: the proteoliposomes yielded a single band with an apparent density of 1.035 g/mL. By subjecting solubilized brush border membrane vesicles (BBMV) to gel filtration on Sephadex G-200 prior to reconstitution, a 7-fold enrichment of the sterol transport activity was achieved relative to the original BBMV. The experimental evidence presented lends strong support to the notion that the sterol transport protein is an integral protein of the brush border membrane which is anchored in the lipid bilayer by at least one hydrophobic domain. The active center(s) is (are) exposed to the external side of the membrane. Anchoring of this protein to the lipid bilayer by a glycosylphosphatidylinositol moiety is unlikely. The reconstituted proteoliposomes behaved very similarly to the original BBMV in terms of facilitated sterol uptake. Using these proteoliposomes, a hitherto unknown activity of the brush border membrane was discovered. Long-chain triacylglycerols can be taken up by this membrane as such and need not be hydrolyzed prior to absorption.

(I) There is little discrimination between cholesterol and the plant sterol sitosterol in the uptake at the brush border membrane (BBM). (II) This difference cannot account for the marked discrimination between cholesterol and sitosterol observed in the absorption of these two sterols by the small-intestinal epithelium. (III) This discrimination occurs during intracellular processing involving the esterification and incorporation into lipoprotein particles of the two sterols. This conclusion is based on a comparative study of sterol uptake by brush border membrane vesicles (BBMV) and sterol absorption by Caco-2 cells. (IV) The uptake of sitosterol by the BBM is energy-independent and facilitated in a manner analogous to cholesterol uptake [Thurnhofer, H., & Hauser, H. (1990a) Biochemistry 29, 2142-2148]. (V) The rate of cholesterol and sitosterol uptake by BBMV from both mixed bile salt micelles and small unilamellar vesicles (SUV) as the donor is directly proportional to the sterol content of the donor. (VI) The pseudo-first-order rate constants k1 for sterol uptake from SUV are independent of the sterol content up to 10-20 mol %. Above that, competition between the two sterols leads to a reduction of the k1 values.

This paper provides unambiguous evidence that brush border membrane vesicles (BBMV) routinely prepared from rabbit small intestine contain a protein that catalyzes the absorption of long-chain cholesteryl ester and ether. The protein is located on the lumenal side of the brush border membrane. The experiments demonstrate that cholesteryl oleate need not be hydrolyzed prior to its incorporation in the BBMV. Unexpectedly and surprisingly, the absorption kinetics of free and esterified cholesterol are very similar in small intestinal BBMV using mixed bile salt micelles and small unilamellar phospholipid vesicles as the donor. The water-soluble form of the protein responsible for this effect is released into the supernatant, probably by autoproteolysis, and catalyzes the exchange of both free and esterified cholesterol between two populations of small unilamellar phospholipid vesicles (SUV). The water-soluble form of the protein was partially purified by a two-step procedure involving gel filtration on Sephadex G-75 and anion-exchange chromatography on Mono Q, yielding a 50-fold increase in the specific activity of the protein. The resulting protein gave two bands on sodium dodecyl sulfate--10% polyacrylamide gel electrophoresis and was used to raise polyclonal antibodies in sheep. The IgG fraction of the sheep antisera blocked the cholesteryl oleate and cholesterol exchange between two populations of SUV mediated by the antigen. The same IgG fraction produced a partial inhibition of cholesterol absorption in small intestinal BBMV. We conclude from the data presented that, contrary to the general belief prevailing in the field of lipid digestion and absorption, long-chain cholesteryl esters may be taken up by the brush border membrane as such and need not be hydrolyzed prior to absorption. The actual contribution of this mechanism to the total absorption of long-chain cholesteryl esters is probably limited by the low solubility of these compounds in mixed bile salt micelles and lipid vesicles.

The solubilization of plasma and organelle membranes by diheptanoylphosphatidylcholine (DHPC) has been studied. This short-chain phosphatidylcholine is shown to act as a mild detergent, solubilizing effectively both kinds of membranes at DHPC concentrations of 10-20 mM (0.5-1%). The size of the resulting mixed protein-lipid-DHPC micelles ranges between 5 and 8 nm. The protein conformation and hence the enzymatic activity are well preserved over a rather large DHPC concentration range (up to 4-5 times the DHPC concentration required for solubilizing the membranes). Evidence is presented that short-chain phosphatidylcholines are superior to most detergents commonly used by biochemists. This is true not only regarding its excellent dispersing power on both phospholipid bilayers (Gabriel & Roberts, 1986) and biological membranes but also as to its capacity to preserve the native protein structure and hence enzymatic activity in the solubilized state. Due to its special properties DHPC lends itself very well not only to membrane solubilization but also to the purification of the solubilized membrane proteins and reconstitution of the proteins into simple lipid bilayers. Concerning the mechanism of membrane solubilization, evidence indicates that DHPC interacts primarily with the lipid bilayer of the membrane and not with the membrane proteins. DHPC solubilizes membranes by being distributed into the lipid bilayer and breaking it up. In the resulting small mixed micelles, the protein remains associated with its preferred intrinsic membrane lipids and is thus stabilized. The protein-intrinsic lipid complex is successfully shielded from unfavorable contacts with H2O by DHPC-intrinsic lipid interactions.

The absorption of monoacylglycerol by small intestinal brush border membrane is a passive process, i.e., the movement of monoacylglycerol from small unilamellar phospholipid vesicles as donor particles through the aqueous medium and the incorporation into the outer monolayer of the lipid bilayer of the brush border membrane are passive processes involving diffusion of the lipid along a concentration gradient. Small unilamellar vesicles of egg phosphatidylcholine containing 1 mol% of radiolabeled hexadecylglycerol were used as donor, and rabbit small intestinal brush border membrane vesicles or intact enterocytes isolated from pig jejunum, as acceptor. Hexadecylglycerol was employed as a lipase-resistant model compound for monoacylglycerols. Both acceptor membranes behave similarly in terms of hexadecylglycerol absorption: the kinetics of hexadecylglycerol absorption are biphasic. The initial fast phase is due to the movement of hexadecylglycerol from the donor particle through the aqueous medium to the outer lipid monolayer of the acceptor membrane, and the second slow phase probably involves the flip-flop motion of hexadecylglycerol from the outer to the inner monolayer of the acceptor membrane. The values for the pseudo-first-order rate constants of the initial fast phase for hexadecylglycerol absorption are relatively large and primarily determined by the high solubility (cmc) of hexadecylglycerol in aqueous media. The pseudo-first-order rate constants depend linearly on the protein (lipid) concentration of the acceptor membrane, indicating that the on rate of the hexadecylglycerol into the brush border membrane is rate limiting. The mechanism of the hexadecylglycerol absorption involves mainly monomer diffusion and probably collision-induced transfer.

The 13C labeled lipid 1[1'-13C]DPPS-NH4+ and its metal salts were used to unambiguously assign all carbonyl vibrations in the infrared spectrum of phosphatidylserines. It is shown that the C=O stretching band at 1741 cm-1 of phosphatidylserines previously assigned to the sn-1 C = O vibration contains contributions from both the sn-1 and the sn-2 carbonyls. The C=O stretching band at frequencies between 1715 and 1730 cm-1 previously assigned to the sn-2 C=O vibration also contains contributions from both carbonyl groups. The frequency dependence observed with the ester carbonyls primarily reflects hydrogen bonding and the polarity of the immediate vicinity. Conformational changes are accounted for in terms of frequency shifts if the conformational change involves the disposition of the C=O groups and in turn the hydrogen bonding properties. The infrared spectra of phospholipids dispersed in aqueous medium in the liquid crystalline state are inconsistent with a simple phospholipid conformation, e.g., with a conformation as found in the single-crystal structure of 1,2-dimyristoyl-sn-phosphatidylcholine and 1,2-dilauroyl-rac-phosphatidylethanolamine. The spectra support the hypothesis proposed earlier (Hauser et al., Biochemistry, 1988) on the basis of existing single-crystal phospholipid structures and NMR evidence. The hypothesis states that several conformations are present in liquid crystalline phospholipid dispersions.(ABSTRACT TRUNCATED AT 250 WORDS)

All classes of phospholipids present in brush border membrane are exchanged in a 1:1 ratio for egg phosphatidylcholine when brush border membrane vesicles from rabbit small intestine are incubated with small unilamellar vesicles of egg phosphatidylcholine. The exchange reaction exhibits biphasic kinetics similar to those of the hydrolysis of brush border membrane phospholipids by phospholipase A2 and sphingomyelinase C. In both reactions there is an initial fast phase followed by a markedly slower one. The phospholipid exchange appears to be catalyzed by intrinsic brush border membrane protein(s), while the digestion by phospholipases is mediated by externally added enzymes. From a comparison of the kinetics of phospholipid exchange and phospholipid hydrolysis, the following conclusions can be drawn: Both sets of experiments indicate the presence of two phospholipid pools differing in the rate of phospholipid exchange and hydrolysis. Except for sphingomyelin, the size of the two phospholipid pools derived from phospholipid exchange is in good agreement with that derived from phospholipid hydrolysis. This is the main finding of this work, and on the basis of this result the two lipid pools are tentatively assigned to phospholipid molecules located on the outer and inner layer of the brush border membrane. The slow rate of phospholipid exchange reflects the rate of transverse or flip-flop movement of phospholipids. The half-time of this motion is approximately 8 h for isoelectric (neutral) phospholipids such as phosphatidylethanolamine and approximately 80 h for negatively charged phosphatidylserine and phosphatidylinositol. Isoelectric phospholipids (phosphatidylcholine, phosphatidylethanolamine) are preferentially located on the inner (cytoplasmic) side (to about 70%) while the negatively charged phospholipids are more evenly distributed: 55-60% are located on the inner side.

31P NMR and infrared spectroscopic methods have been used to study the formation of small unilamellar vesicles by the pH-jump method. It is shown that increasing the pH of different lamellar phospholipid dispersions (phosphatidic acids and phosphatidylserines) induces a pH gradient. This pH gradient is estimated to be 4 +/- 1 pH units, and its direction is such that the inner monolayer of the vesicles is at lower pH. There is spectroscopic evidence for tighter packing of the lipid hydrocarbon chains in the inner monolayer, probably due to the constraints imposed by the high curvature of the small vesicles formed. These results are discussed in terms of the driving force of the spontaneous vesiculation.

Absorption of cholesterol by small intestinal brush border membrane from either mixed micelles or small unilamellar vesicles is protein-mediated. It is a second-order reaction. The kinetic data are consistent with a mechanism involving collision-induced transfer of cholesterol. With micelles as the donor particle, there is net transfer of cholesterol while with small unilamellar vesicles as the donor, cholesterol is evenly distributed between the two lipid pools at equilibrium. The cholesterol absorption by brush border membrane from both mixed micelles and small unilamellar vesicles reveals saturation kinetics. Proteolytic treatment of brush border membrane with papain releases about 25% of the total membrane protein. As a result, the cholesterol uptake by brush border membrane changes from a second-order reaction to a first-order one. The reaction mechanism changes from collision-induced cholesterol uptake to a mechanism involving diffusion of monomeric cholesterol through the aqueous phase. The protein(s) released into the supernatant by papain treatment of brush border membrane exhibit(s) cholesterol exchange activity between two populations of small unilamellar vesicles. The supernate-protein(s) bind(s) the spin-labeled cholesterol analogue 3-doxyl-5 alpha-cholestane.

The temperature dependence of the packing (order) and fluidity (microviscosity) of rabbit small, intestinal brush border vesicle membranes and of liposomes made from their extracted lipids has been investigated by using a variety of lipid spin probes. The lipids in the brush border membrane are present essentially as a bilayer. Compared to other mammalian membranes, the brush border membrane appears to be characterized by a relatively high packing order as well as microviscosity. At body temperature, the lipid molecules undergo rapid, anisotropic motion, which is essentially a fast rotation about an axis approximately perpendicular to the bilayer normal. Both the order (motional anisotropy) and the microviscosity increase with decreasing temperature and with increasing distance from the center of the bilayer. Qualitatively similar motional or fluidity gradients have been reported for other mammalian and bacterial membranes. The liposomes made from the extracted lipids have a somewhat lower packing order and a slightly higher fluidity than brush border vesicle membranes. The differences are, however, small indicating that the packing and the fluidity (microviscosity) of the membrane are primarily determined by the lipid composition. Membrane-associated proteins and cytoskeleton cannot play a dominant role in determining the order and fluidity of the lipid bilayer. Discontinuities are observed in the temperature dependence of various spectral parameters, the order parameter S, the rotational correlation time tau, and 2,2,6,6-tetramethylpiperidinyloxy partitioning. They are assigned to phase transitions and/or phase separations of the membrane lipids. These discontinuities occur at about 30, 20, and 13 degrees C for 5-doxyl-, 12-doxyl-, and 16-doxylstearic acid, respectively. The apparent transition temperature depends on the location of the spin probe along the bilayer normal, being higher the closer the probe is to the membrane surface. This indicates the possibility that chain melting is progressive and spreads with increasing temperature from the center of the membrane outward.

The thermotropic phase behavior of fully hydrated Na+ and/or NH4+ salts of 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS) was determined by temperature-dependent infrared spectra. The molecular level properties and thermal phase behavior of DMPS-Li+ complexes were also characterized by infrared spectroscopy. With increasing concentrations of Li+, the infrared spectra reveal the appearance of a second, more ordered, lipid phase which shows a gel to liquid-crystal transition at significantly higher temperatures (75-95 degrees C) than the Na+ or NH4+ salts of DMPS (39 degrees C). Li+ binds to the phosphate and carboxylate groups of DMPS, resulting in the following changes: (1) water of hydration is lost from both the carboxylate and phosphate groups; (2) there are changes in the conformation of the glycerol backbone but not in the P-O ester bonds of the phosphate group which remain in the gauche-gauche conformation; and (3) the packing of the fatty acyl chains becomes more ordered. In addition, the properties of the DMPS-Ca2+ complex were studied by infrared spectroscopy. While the DMPS-Ca2+ complex is also characterized by rigidly packed, well-ordered fatty acyl chains, the mode of Ca2+ binding to the DMPS head groups differs significantly from that of Li+ binding. By comparison, with dry DMPS-Ca2+ [Casal, H. L., Mantsch, H. H., Paltauf, F., & Hauser, H. (1987) Biochim. Biophys. Acta (in press)], the phosphate group undergoes a conformational change, probably to the antiplanar-antiplanar conformation, and loses its water of hydration.(ABSTRACT TRUNCATED AT 250 WORDS)

The structure and thermotropic properties of a homologous series of diacylphosphatidylserines (PS) in the anhydrous and hydrated states have been examined with low-angle X-ray diffraction and differential scanning calorimetry. In the anhydrous state at low temperatures both acidic PS and its NH4+ salts exhibit lamellar bilayer crystal forms that transform to liquid-crystalline hexagonal (type II) structures at higher temperatures. The crystal leads to liquid-crystal transition temperature increases with increasing chain length, the transition temperature of an NH4+-PS being higher than that of its corresponding acidic form. In contrast, the transition enthalpies of the acidic PS are higher than those of the NH4+ salt forms. Hydrated cyclic PS and NH4+-PS exhibit reversible lamellar gel leads to liquid-crystal transitions. In this case the acidic form undergoes this chain length dependent transition at a higher temperature, but with a lower enthalpy change, than the NH4+-PS. Both below and above the hydrocarbon chain melting transition, hydrated lamellar bilayer structures are present. The temperature-composition phase diagram of the NH4+-dimyristoyl-PS/H2O system has been studied in detail. The chain melting transition decreases with increasing hydration, reaching a limiting value of 39 degrees C. X-ray diffraction shows that both the bilayer gel structure and the bilayer liquid-crystal form take up water continuously (i.e., no hydration limit), a characteristic of lipid bilayers with a net charge. Electron-density profiles a NH4+-dimyristoyl-PS at different hydration levels permit detailed analysis of the structural parameters of the PS bilayer.

The interaction of divalent cations with a homologous series of diacylphosphatidylserines (diacyl-PS) has been studied by differential scanning calorimetry and X-ray diffraction. Hydrated di-C14-PS (DMPS) exhibits a gel leads to liquid-crystal bilayer transition at 39 degrees C (delta H = 7.2 kcal/mol of DMPS). With increasing MgCl2 concentration, progressive conversion to a phase exhibiting a high melting (98 degrees C), high enthalpy (delta H congruent to 11.0 kcal/mol of DMPS) transition is observed. Similar behavior is observed for DMPS with increasing CaCl2 concentration. In this case, the high-temperature transition of the Ca2+-DMPS complex occurs at approximately 155 degrees C and is immediately followed by an exothermic transition probably associated with PS decomposition. For di-C12-, di-C14-, di-C16- (DPPS), and di-C18-PS, the transition temperatures of the Ca2+-PS complexes are in the range 151-155 degrees C; only di-C10-PS exhibits a significantly lower value, 142 degrees C. A different pattern of behavior is exhibited by DPPS in the presence of Sr2+ or Ba2+, with transitions in the range 70-80 degrees C being observed. X-ray diffraction of the Ca2+-PS complexes at 20 degrees C provides evidence of structural homology. All Ca2+-PS complexes exhibit bilayer structures, the bilayer periodicity increasing linearly from 35.0 A for di-C10-PS to 52.5 A for di-C18-PS. Wide-angle X-ray diffraction data indicate that hydrocarbon chain "crystallization" occurs on Ca2+-PS complex formation.(ABSTRACT TRUNCATED AT 250 WORDS)

A combination of surface monolayer, scanning calorimetry, 31P NMR, and spin-label ESR techniques has been used to monitor the interactions of monovalent (NH4+, Na+, and Li+) and divalent (Ca2+) cations with phosphatidylserines (PS) differing in their levels of chain unsaturation. Comparisons are made between the disaturated dimyristoyl-, dipalmitoyl-, and dihexadecyl-PS (DMPS, DPPS, and DHPS), saturated cis-monounsaturated palmitoyloleoyl-PS (POPS) (and bovine brain PS), di-trans-monounsaturated dielaidoyl-PS (DEPS), and di-cis-monounsaturated dioleoyl-PS (DOPS). Na+ and NH4+ cations interact weakly with all PS monolayers and bilayers without significant changes in molecular conformation, chain packing, or headgroup dynamics and without dependence on chain composition. In contrast, considering these structural and dynamic parameters, Li+ shows a gradation in its interaction with PS (DMPS greater than POPS approximately bovine brain PS greater than DOPS), suggesting that Li+-PS interactions depend on the interfacial properties of the PS molecules (e.g., surface area). Finally, Ca2+ interacts strongly with all PS monolayers and bilayers, without obvious chain selectivity. Thus, ion binding to PS depends not only on the properties of the cation (Na+ vs Li+ vs Ca2+) but also on the molecular details of the PS membrane surface.

Lipids in the plasma membrane of the general fatty acid auxotroph Butyrivibrio S2 pack as a bilayer that is characterized by a high order and high motional anisotropy and a low membrane fluidity compared to mammalian plasma membranes. Lipid packing as determined by the electron spin resonance (ESR) order parameter and membrane fluidity as measured by ESR correlation times are, however, comparable to those of other bacterial membranes. Membranes of the organism grown with saturated fatty acids of well-defined hydrocarbon chain length undergo a broad reversible endothermic phase transition, the peak temperature of which is well below the growth temperature; the end-point temperature of this thermal transition approximately coincides with the minimum temperature supporting significant growth of the organism. The lipid phase transition is also reflected in the temperature dependence of various ESR parameters, whereby the transition temperature thus derived is higher than the peak temperature of the endothermic transition but still lower than the growth temperature. ESR and calorimetry evidence taken together suggest that the endothermic transition is a gel to liquid-crystal transition and that, at the growth temperature, the plasma membrane of Butyrivibrio S2 is in the liquid-crystalline state. Similar values were measured for the order parameter of cell membranes of Butyrivibrio S2 regardless of whether the organism was grown on myristic, palmitic, or stearic acid. Butyrivibrio S2 has a mechanism enabling it to maintain membrane packing and fluidity at a fairly constant level.(ABSTRACT TRUNCATED AT 250 WORDS)

The total lipids extracted from brush border membranes form smectic lamellar phases when dispersed in water. 31P broad-band nuclear magnetic resonance (NMR) shows that between body temperature (37 degrees C) and freezing of the solvent, the extracted lipids form bilayers with the lipid molecules undergoing fast anisotropic motion. This is also true for the lipids present in the brush border membrane. The electron spin resonance (ESR) results obtained with various hydrophobic spin probes incorporated in either brush border vesicle membranes or their extracted lipids are consistent with this interpretation. By use of a variety of chemically different spin-labels, the temperature dependence of brush border membranes and their extracted lipids was probed. The temperature dependence of various ESR spectral parameters shows discontinuities that, by comparison with differential scanning calorimetry, are assigned to a lipid thermotropic phase transition. Differential scanning calorimetry shows that the lipid in brush border membranes undergoes a broad, reversible phase transition of low enthalpy between 10 and 30 degrees C, with a peak temperature of about 25 degrees C. Hence, the brush border membrane of rabbit small intestine functions in the liquid-crystalline state, well above the peak temperature and also above the upper limit of the lipid phase transition. Therefore, in itself, the thermotropic lipid phase transition is unlikely to play a physiological role. The low enthalpy of the lipid phase transition, indicative of a lack of cooperativity, is primarily attributed to the relatively high cholesterol content and to heterogeneity in the lipid composition of this membrane [Hauser, H., Howell, K., Dawson, R. M. C., & Bowyer, D. E. (1980) Biochim. Biophys. Acta 602, 567-577].(ABSTRACT TRUNCATED AT 250 WORDS)

Spin-spin coupling constants derived from high-resolution 1H NMR spectra of pure 1-myristoyl-sn-glycero-3-phosphocholine (MLPC) micelles and 60:1 mol/mol been analyzed in order to determine the effects of apoprotein on phosphatidylcholine (PC) polar group conformation. The shift ratios of the polar group proton resonances after addition of the paramagnetic shift reagent Fe(CN)6(3-) to the above MLPC systems, egg PC small unilamellar vesicles, and human HDL3 have been used to compare the PC polar group conformations in all systems. The location of the largely alpha-helical apo A-I molecules in the complex with MLPC was deduced from its effects on the chemical shifts and spin-lattice relaxation times (T1) of the well-resolved 1H resonances from the various parts of the lipid molecules. The data are consistent with the apo A-I molecules lying in the surface fo the MLPC micelle with their amphipathic, alpha-helical segments intercalated among the glycerophosphocholine groups of the lipid molecules so that aromatic amino acid side chains are interspersed among the lipid hydrocarbon chains. This leads to a spacing out of the glycerol backbones and immediately adjacent methylene groups of the MLPC molecules, thereby causing an enhancement of the motions affecting T1. The presence of apo A-I at the lipid-water interface apparently does no perturb the PC polar group conformation, indicating that this conformation is determined by intramolecular effects. The preferred conformation of the phosphocholine group (Hauser, H., Pascher, I., Pearson, R. H., & Sundell, S. (1981) Biochim. Biophys, Acta 650, 21-51] is characterized by an almost exclusively gauche conformation of the choline group and predominantly antiperiplanar conformations about the C-C-O-P and P-O-C-C bonds. The PC molecules in MLPC micelles, MLPC-apo A-I complexes, egg PC vesicles, and HDL3 all have this polar group conformation.

A new ESR assay has been developed for the characterization of unilamellar lipid vesicles. It is based on the reduction by photogenerated FADH2 of amphiphilic spin-labels having the spin in the polar group. FADH2 is generated in situ under anaerobic conditions from its oxidized form (FAD) by photoreduction in the presence of excess EDTA as the reducing agent. Photoreduction is induced by exposing the FAD/EDTA mixture to white light of a commercial slide projector. FADH2 as an impermeable agent reduces spin-label molecules located on the outer layer of the bilayer that are readily accessible in a first fast reaction; spin-label located on the inner layer of the bilayer is reduced in a second slow reaction. The ESR assay is suitable for the routine characterization of unilamellar membrane vesicles: it allows the determination of the vesicle size, the entrapped volume, the bilayer asymmetry, the bilayer integrity, and the vesicle stability. The ESR assay developed is of general applicability: it can be used with charged and uncharged bilayers which may be labeled with either neutral or charged spin-labels. An assessment of the new ESR assay is given in comparison to the existing ascorbate method which uses sodium ascorbate as the reducing agent. Various other potential reducing agents for spin-labels have been tested and found unsuitable for the ESR assays discussed here.

Aqueous dispersions of phosphatidic acid and mixtures of phosphatidic acid with other phospholipids vesiculate when the pH is transiently increased to a pH near or above the second pK of the phosphatidic acid. Both small unilamellar vesicles (SUV) of a narrow size distribution (average diameter 25-30 nm) and large unilamellar vesicles (LUV) of a wide size distribution are formed. The fraction of SUV increases linearly with increasing pH from 6 to 12 and was also increased by increasing the rate of the pH change from 2 min to 1 s. For mixed phospholipid dispersions, the fraction of SUV appears to be linearly related to the phosphatidic acid content. Unilamellar vesicles formed by the transient pH increase are subsequently relatively stable to changes in pH and ionic strength. If, after vesiculation is induced, the dispersion (pH 7-8) is acidified to pH 3, the fraction of SUV decreases. However, the multilamellar structures present in the original dispersion do not re-form, and most of the reduction in the SUV fraction is reversible when the pH is returned to 7-8. The addition of NaCl to the dispersion after vesiculation has no effect on the fraction of SUV up to physiological NaCl concentrations. Subsequent addition of NaCl to a concentration in excess of 0.2 M reduces the SUV fraction; this disappears above 1 M NaCl due to aggregation or fusion. SUV of phosphatidic acid or mixed phospholipids containing phosphatidic acid can be stored at 4 degrees C for 14 days without detectable aggregation and/or fusion.

Hemoglobin is encapsulated in liposomes of different lipid composition. The resulting dispersion consists primarily of multilamellar liposomes (hemosomes) of a wide particle size distribution (diameter ranging mainly between 0.1 and 1 micron). The encapsulation efficiency is significantly larger with liposomes containing negatively charged lipids as compared to liposomes made of phosphatidylcholine. The integrity of the phospholipid bilayer is maintained in the presence of hemoglobin. The reaction rate of CO binding to encapsulated hemoglobin is reduced compared to that of free hemoglobin, but it is still greater than that observed in red blood cells. Hemoglobin encapsulated in liposomes made from negatively charged phospholipids is less stable than hemoglobin entrapped in isoelectric phosphatidylcholine. The instability of hemoglobin is due to the protein interacting with the negatively charged lipid bilayer. This interaction leads in turn to hemoglobin denaturation, possibly involving the dissociation of the heme group from the heme-globin complex. The nature of the negatively charged phospholipid is important in promoting the interaction with hemoglobin, the effect being in the order phosphatidic acid greater than phosphatidylinositol congruent to phosphatidylglycerol greater than phosphatidylserine. The presence of equimolar amounts of cholesterol in the phospholipid bilayer has a stabilizing effect on hemoglobin. This effect is pronounced with saturated phospholipids, but it is also observed, though to a lesser extent, with unsaturated ones, indicating that the bilayer fluidity has a modulating effect. The presence of cholesterol possibly interferes with secondary interactions following the binding of hemoglobin to the negatively charged lipid bilayer.

The monolayer properties and thermal behavior of different phosphatidylserines are presented. At neutral pH and 22 degrees C, saturated phosphatidylserines form condensed monolayers while unsaturated phosphatidylserines form liquid-expanded films. Under similar conditions, dimyristoylphosphatidylserine undergoes a transition from the liquid-expanded to the condensed state. At pH 4 and 22 degrees C, the surface pressure-area isotherms are shifted to smaller areas relative to the monolayers recorded at neutral pH. The condensation observed at pH 4 is close to that produced at pH 7.4 by the addition of 10 mM CaCl2. As regards the molecular packing in monolayers and the thermal behavior, 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS) and its ether analogue are similar, albeit not identical. Below 30 mN/m, monolayers of the ether analogue are even more condensed than those of DPPS. The order-disorder transition of the ether analogue occurs usually at higher temperatures than that of the diacyl compound. Sonicated phosphatidylserine dispersions consisting of small unilamellar vesicles show anomalous thermal properties compared to sonicated phosphatidylcholine dispersions. They exhibit sharp order-disorder transitions at similar or even slightly elevated temperatures compared to unsonicated phosphatidylserine dispersions. This anomaly is explained in terms of a pH gradient across the bilayer membrane of the small unilamellar phosphatidylserine vesicle. The internal surface pH is more acidic than the external pH, leading to some protonation of phosphatidylserine molecules. This in turn leads to a condensation of phosphatidylserine molecules on the inner bilayer surface. Such a gradient is proposed to be responsible for the thermodynamic stability of highly curved negatively charged bilayer vesicles.

Infrared spectroscopy has been used to characterize the thermal-phase behavior of fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) as well as their interaction with Li+ and Ca2+. The order-disorder transition of POPS-NH4+ is at 17 degrees C; in the presence of Li+ a POPS-Li+ complex is formed, and the transition temperature of this complex is 40 degrees C. DOPS-NH4+ has an order-disorder transition at -11 degrees C, and unlike POPS the addition of Li+ has no effect on the thermal behavior of DOPS-NH4+. This indicates that the binding of Li+ to DOPS is negligible or very weak. Li+ binds to the phosphate and carboxylate groups of POPS, and as a result these groups lose their water of hydration. Li+ binding induces a conformational change, probably in the glycerol backbone of POPS; however, the conformation of the two P-O ester bonds remains gauche-gauche as in POPS-NH4+. Both POPS and DOPS form crystalline complexes with Ca2+. As a result of Ca2+ binding to the phosphate, this group loses its water of hydration and there is a conformational change in the P-O ester bonds from gauche-gauche to antiplanar-antiplanar. In contrast to the POPS-Li+ complex, the carboxylate group remains hydrated in the Ca2+ complexes. Furthermore, in these PS-Ca2+ complexes a new hydrogen bond is formed between one of the ester C=O groups and probably water. Such a situation is not found in the NH4+ and Li+ salts of phosphatidylserine.

The swelling properties of lipid mixtures consisting of phosphatidylcholine and a charged single-chain detergent have been studied. The work presented here is confined to lipid mixtures forming smectic lamellar phases in H2O. These mixtures exhibit continuous swelling with increasing water content, provided the surface charge density exceeds a threshold value of about 1-2 microC/cm2. In excess H2O, such mixtures undergo spontaneous vesiculation: unilamellar vesicles form spontaneously when excess H2O or salt solutions of moderate ionic strength (I less than 0.2) are added to the dried film of such lipid mixtures. The resulting dispersion of unilamellar vesicles is usually polydisperse. Its average size depends on the detergent/phospholipid mole ratio, decreasing with increasing detergent content. It is shown that in the phase diagram of three-component systems consisting of phosphatidylcholine, a charged single-chain detergent, and excess H2O there is a compositional range, though narrow, within which the small unilamellar vesicle (diameter less than 100 nm) is the thermodynamically most stable structure. This behavior is characteristic of charged, single-chain detergents of 14 and more C atoms. Many pharmacologically active compounds are amphiphilic and surface-active, and as such, they will orient at phospholipid-water interfaces, imparting a net surface charge to neutral lipid surfaces. It is shown that such drugs exhibit detergent-like action. Mixed films of phosphatidylcholine and a pharmacologically active compound behave similarly to phosphatidylcholine-detergent mixtures: they undergo spontaneous vesiculation when excess H2O or salt solutions of moderate ionic strength are added. In this case, the drug itself induces vesiculation; possible pharmacological implications of this finding are discussed.

Hemoglobin (Hb) was incubated with small unilamellar vesicles of different lipid compositions in physiological or low pH-low ionic strength media, and the alterations in the protein and bilayer structure were analyzed by measuring (i) the sedimentation properties and elution pattern of the vesicles upon gel filtration, (ii) the quenching effect of Hb on the fluorescence of membrane-embedded probes, (iii) the intrinsic fluorescence of Hb, and (iv) the Soret spectra of Hb. The results indicate complex formation between Hb and the membrane, followed by intercalation of the protein into the bilayer. These processes can lead to secondary alterations, including aggregation, peroxidative decomposition of unsaturated fatty acids, unfolding of Hb, oxidation of the heme iron, displacement of the heme relative to globin, and oxidative or nonoxidative deconjugation of the porphyrin ring. Complex formation and intercalation of Hb into the bilayer are primarily due to hydrophobic interaction between the protein and the membrane, whereas displacement of the heme and its nonoxidative deconjugation are elicited by ionic interaction between the heme and negative membrane surface charges. These charges, on the other hand, inhibit the oxidative processes in unsaturated lipid vesicles. The rate and extent of alterations both in the protein and in the membrane show inverse correlation with the stability of the heme-globin linkage. The liquid-crystalline phase state promotes the penetration of Hb into the bilayer, whereas displacement of the heme is most expressed in the case of saturated bilayers in the gel state. Cholesterol inhibits both the hydrophobic and ionic interactions between the protein and bilayer, most remarkably the partition of the heme into negatively charged saturated bilayers. It is suggested that the effects of cholesterol are mediated through changes in membrane fluidity.

Cytochrome P-450 and NADPH-cytochrome P-450 reductase were reconstituted in unilamellar lipid vesicles prepared by the cholate dialysis technique from pure dimyristoylphosphatidylcholine (DMPC), pure dipalmitoylphosphatidylcholine (DPPC), pure dioleoylphosphatidylcholine (DOPC), and phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine (PC/PE/PS) (10:5:1). As probes for the vesicles' hydrocarbon region, 1,6-diphenyl-1,3,5-hexatriene (DPH) and spin-labeled PC were used. The steady-state and time-resolved fluorescence parameters of DPH were determined as a function of temperature and composition of liposomes. Incorporation of either protein alone or together increased the steady-state fluorescence anisotropy (rs) of DPH in DOPC and PC/PE/PS (10:5:1) liposomes. In DMPC and DPPC vesicles, the proteins decreased rs significantly below the transition temperature (Tc) of the gel to liquid-crystalline phase transition. Time-resolved fluorescence measurements of DPH performed in reconstituted PC/PE/PS and DMPC proteoliposomes showed that the proteins disorder the bilayer both in the gel and in the liquid-crystalline phase. Little disordering by the proteins was observed by a spin-label located near the mid-zone of the bilayer 1-palmitoyl-2-(5-doxylstearoyl)-3-sn-phosphatidylcholine (8-doxyl-PC), whereas pronounced disordering was detected by 1-palmitoyl-2-(8-doxylpalmitoyl)-3-sn-phosphatidylcholine (5-doxyl-PC), which probes the lipid zone closer to the polar part of the membrane. Fluorescence lifetime measurements of DPH indicate an average distance of greater than or equal to 60 A between the heme of cytochrome P-450 and DPH.

The kinetics of lipid transfer from small unilamellar vesicles as the donor to brush border vesicles as the acceptor have been investigated by following the transfer of radiolabeled or spin-labeled lipid molecules in the absence of exchange protein. The labeled lipid molecules studied were various radiolabeled and spin-labeled phosphatidylcholines, radiolabeled cholesteryl oleate, and a spin-labeled cholestane. At a given temperature and brush border vesicle concentration similar pseudo-first-order rate constants (half-lifetimes) were observed for different lipid labels used. The lipid transfer is shown to be an exchange reaction leading to an equal distribution of label in donor and acceptor vesicles at equilibrium (time t----infinity). The lipid exchange is a second-order reaction with rate constants being directly proportional to the brush border vesicle concentration. The results are only consistent with a collision-induced exchange of lipid molecules between small unilamellar phospholipid vesicles and brush border vesicles. Other mechanisms such as collision-induced fusion or diffusion of lipid monomers through the aqueous phase are negligible at least under our experimental conditions.

The conformation of the glycerol group of a number of diacyl and monoacyl (lyso) phospholipids differing in the chemical nature of the head group was studied by 1H high-resolution NMR and X-ray crystallography. The NMR measurements were carried out with solutions or micellar dispersions of the lipids in deuteriated organic solvents or 2H2O. Both solutions, in which the lipid is present as monomers, and lipid micelles give rise to good high-resolution NMR spectra exhibiting spin coupling hyperfine interactions. From 1H spin coupling it is concluded that there are two stable conformations about the glycerol C(2)-C(3) bond of phospholipids. One of these (rotamer A) is characterized by torsion angles theta 3 = antiperiplanar, theta 4 = +synclinal, and the other (rotamer B) by theta 3 = +synclinal, theta 4 = -synclinal. In both rotamers A and B the ester oxygens on the glycerol carbon atoms C(2) and C(3) are synclinal, and hence both types of rotamers readily allow the parallel alignment of the two hydrocarbon chains. By comparison of NMR and single-crystal X-ray data it is obvious that both conformations are minimum free energy conformations. Rotamer A is the conformation prevailing in phospholipid single-crystal structures. The conformation of rotamer B is also found in phospholipid single-crystal structures though to a lesser extent, e.g., in 2,3-dilauroyl-DL-glycero-1-phospho-N,N-dimethylethanolamine and 2,3-dimyristoyl-D-glycero-phospho-DL-glycerol. NMR measurements indicate that in liquid crystals the diacylglycerol part of phospholipids fluctuates between the two stable staggered conformations of rotamers A and B.(ABSTRACT TRUNCATED AT 250 WORDS)