Anthroyloxy Fatty Acids Research Articles

Depth-Dependent Heterogeneity in Membranes by Fluorescence Lifetime Distribution Analysis.

Biological membranes display considerable anisotropy due to differences in composition, physical characteristics, and packing of membrane components. In this Letter, we have demonstrated the environmental heterogeneity along the bilayer normal in a depth-dependent manner using a number of anthroyloxy fatty acid probes. We employed fluorescence lifetime distribution analysis utilizing the maximum entropy method (MEM) to assess heterogeneity. Our results show that the fluorescence lifetime heterogeneity varies considerably depending on fluorophore location along the membrane normal (depth), and it is the result of the anisotropic environmental heterogeneity along the bilayer normal. Environmental heterogeneity is reduced as the reporter group is moved from the membrane interface to a deeper hydrocarbon region. To the best of our knowledge, our results constitute the first experimental demonstration of anisotropic heterogeneity in bilayers. We conclude that such graded environmental heterogeneity represents an intrinsic characteristics of the membrane bilayer and envisage that it has a role in the conformation and orientation of membrane proteins and their function.

Fatty Acid Transfer from Yarrowia lipolytica Sterol Carrier Protein 2 to Phospholipid Membranes

Sterol carrier protein 2 (SCP2) is an intracellular protein domain found in all forms of life. It was originally identified as a sterol transfer protein, but was recently shown to also bind phospholipids, fatty acids, and fatty-acyl-CoA with high affinity. Based on studies carried out in higher eukaryotes, it is believed that SCP2 targets its ligands to compartmentalized intracellular pools and participates in lipid traffic, signaling, and metabolism. However, the biological functions of SCP2 are incompletely characterized and may be different in microorganisms. Herein, we demonstrate the preferential localization of SCP2 of Yarrowia lipolytica (YLSCP2) in peroxisome-enriched fractions and examine the rate and mechanism of transfer of anthroyloxy fatty acid from YLSCP2 to a variety of phospholipid membranes using a fluorescence resonance energy transfer assay. The results show that fatty acids are transferred by a collision-mediated mechanism, and that negative charges on the membrane surface are important for establishing a “collisional complex”. Phospholipids, which are major constituents of peroxisome and mitochondria, induce special effects on the rates of transfer. In conclusion, YLSCP2 may function as a fatty acid transporter with some degree of specificity, and probably diverts fatty acids to the peroxisomal metabolism.

Fatty acid transfer from intestinal fatty acid binding protein to membranes: electrostatic and hydrophobic interactions

Intestinal fatty acid binding protein (IFABP) is thought to participate in the intracellular transport of fatty acids (FAs). Fatty acid transfer from IFABP to phospholipid membranes is proposed to occur during protein-membrane collisional interactions. In this study, we analyzed the participation of electrostatic and hydrophobic interactions in the collisional mechanism of FA transfer from IFABP to membranes. Using a fluorescence resonance energy transfer assay, we examined the rate and mechanism of transfer of anthroyloxy-fatty acid analogs a) from IFABP to phospholipid membranes of different composition; b) from chemically modified IFABPs, in which the acetylation of surface lysine residues eliminated positive surface charges; and c) as a function of ionic strength. The results show clearly that negative charges on the membrane surface and positive charges on the protein surface are important for establishing the "collisional complex", during which fatty acid transfer occurs. In addition, changes in the hydrophobicity of the protein surface, as well as the hydrophobic volume of the acceptor vesicles, also influenced the rate of fatty acid transfer. Thus, ionic interactions between IFABP and membranes appear to play a primary role in the process of fatty acid transfer to membranes, and hydrophobic interactions can also modulate the rates of ligand transfer.

Localization of bilirubin in phospholipid bilayers by parallax analysis of fluorescence quenching

It has been proposed that the neurotoxicity observed in severely jaundiced infants results from the binding of unconjugated bilirubin to nerve cell membranes. However, despite potentially important clinical ramifications, there remains significant controversy regarding the physical nature of bilirubin-membrane interactions. We used the technique of parallax analysis of fluorescence quenching (Chattopadhyay, A., and E. London. 1987. Biochemistry. 26: 39–45) to measure the depth of penetration of bilirubin in model phospholipid bilayers. The localization of unconjugated bilirubin and ditaurobilirubin within small unilamellar vesicles composed of dioleoylphosphatidylcholine was determined through an analysis of the quenching of bilirubin fluorescence by spin-labeled phospholipids, and by bilirubin-mediated quenching of a series of anthroyloxy fatty acid probes at various depths within the membrane bilayer. Findings were further verified with potassium iodide as an aqueous quencher. Our results indicate that, at pH 10, unconjugated bilirubin localizes approximately 20 Å from the bilayer center, in the region of the polar head groups. Further analyses suggest a modest influence of pH, membrane cholesterol content, and vesicle diameter on the bilirubin penetration depth. Taken together, these data support that, under physiologic conditions, bilirubin localizes to the polar region of phospholipid bilayers, near the membrane-water interface. —Zucker, S. D., W. Goessling, E. J. Bootle, and C. Sterritt. Localization of bilirubin in phospholipid bilayers by parallax analysis of fluorescence quenching.

Consequences of ω-6-Oleate Desaturase Deficiency on Lipid Dynamics and Functional Properties of Mitochondrial Membranes ofArabidopsis thaliana

We probed the role of the polyunsaturated fatty acids on the dynamic and functional properties of mitochondrial membranes using the fad2 mutant of Arabidopsis thaliana, deficient in omega-6-oleate desaturase. In mitochondria of this mutant, the oleic acid content exceeded 70% of the total fatty acids, and the lipid/protein ratio was greatly enhanced. As a consequence, local microviscosity, probed by anthroyloxy fatty acid derivatives, was increased by 30%, whereas the lipid lateral diffusion, assayed using 1-pyrenedodecanoic acid, was approximately 4 times increased. Functional parameters such as oxygen consumption rate under phosphorylating and nonphosphorylating conditions and proton permeability of the inner mitochondrial membrane were significantly reduced in fad2 mitochondrial membranes, while the thermal dependence of the respiration was enhanced. Moreover, metabolic control analysis of the respiration clearly showed an enhancement of the control exerted by the membrane proton leaks. Our data suggest that the loss of omega-6-oleate desaturase activity in Arabidopsis cells induced an enhancement of both microviscosity and lipid/protein ratio of mitochondrial membranes, which in turn were responsible for the change in lateral mobility of lipids and for bioenergetic parameter modifications.

Lipid lateral diffusion and local microviscosity in plant mitochondrial membranes with various length and unsaturation of fatty acids

Two main aspects of the lipid dynamics, local microviscosity and lateral diffusion, were investigated in intact plant mitochondria isolated from different tissues exhibiting large differences in their fatty acids in terms of unsaturation (amount of linoleic and linolenic acids) or length of the hydrocarbon chains. In addition, the same parameters were determined in the outer and inner membranes isolated from cauliflower mitochondria, which differed not only in the fatty acid composition but also by the lipid-to-protein ratio. In intact mitochondria, local microviscosity assayed with anthroyloxy-fatty acids exhibited a transverse gradient from the surface to the center of the bilayer, which was mainly affected by the unsaturation index and the content in linoleic or linolenic acids. In contrast, lipid lateral diffusion increased as the content in linolenic or palmitic acids increased, but was not directly correlated to the unsaturation index. Interestingly, local microviscosity at the membrane surface was higher in the outer membrane than in the inner membrane, whereas no significant difference was found in lipid lateral diffusion. These results indicate that the influence of the fatty acid composition of mitochondrial membranes on the dynamics of the phospholipid bilayer depends on the type of movement considered and suggest that other parameters, such as the protein content of the bilayer, also affect membrane fluidity.

Lipid lateral diffusion and local microviscosity in plant mitochondrial membranes with various length and unsaturation of fatty acids

Two main aspects of the lipid dynamics, local microviscosity and lateral diffusion, were investigated in intact plant mitochondria isolated from different tissues exhibiting large differences in their fatty acids in terms of unsaturation (amount of linoleic and linolenic acids) or length of the hydrocarbon chains. In addition, the same parameters were determined in the outer and inner membranes isolated from cauliflower mitochondria, which differed not only in the fatty acid composition but also by the lipid-to-protein ratio. In intact mitochondria, local microviscosity assayed with anthroyloxy-fatty acids exhibited a transverse gradient from the surface to the center of the bilayer, which was mainly affected by the unsaturation index and the content in linoleic or linolenic acids. In contrast, lipid lateral diffusion increased as the content in linolenic or palmitic acids increased, but was not directly correlated to the unsaturation index. Interestingly, local microviscosity at the membrane surface was higher in the outer membrane than in the inner membrane, whereas no significant difference was found in lipid lateral diffusion. These results indicate that the influence of the fatty acid composition of mitochondrial membranes on the dynamics of the phospholipid bilayer depends on the type of movement considered and suggest that other parameters, such as the protein content of the bilayer, also affect membrane fluidity.

The helical domain of intestinal fatty acid binding protein is critical for collisional transfer of fatty acids to phospholipid membranes.

Fatty acid binding proteins (FABPs) exhibit a beta-barrel topology, comprising 10 antiparallel beta-sheets capped by two short alpha-helical segments. Previous studies suggested that fatty acid transfer from several FABPs occurs during interaction between the protein and the acceptor membrane, and that the helical domain of the FABPs plays an important role in this process. In this study, we employed a helix-less variant of intestinal FABP (IFABP-HL) and examined the rate and mechanism of transfer of fluorescent anthroyloxy fatty acids (AOFA) from this protein to model membranes in comparison to the wild type (wIFABP). In marked contrast to wIFABP, IFABP-HL does not show significant modification of the AOFA transfer rate as a function of either the concentration or the composition of the acceptor membranes. These results suggest that the transfer of fatty acids from IFABP-HL occurs by an aqueous diffusion-mediated process, i.e., in the absence of the helical domain, effective collisional transfer of fatty acids to membranes does not occur. Binding of wIFABP and IFABP-HL to membranes was directly analyzed by using a cytochrome c competition assay, and it was shown that IFABP-HL was 80% less efficient in preventing cytochrome c from binding to membranes than the native IFABP. Collectively, these results indicate that the alpha-helical region of IFABP is involved in membrane interactions and thus plays a critical role in the collisional mechanism of fatty acid transfer from IFABP to phospholipid membranes.

Flip-flop is slow and rate limiting for the movement of long chain anthroyloxy fatty acids across lipid vesicles.

An issue that is central to understanding cellular fatty acid (FA) metabolism is whether physiologic transport of FA across cell membranes requires protein mediation or can be satisfied by the rate of spontaneous movement through the lipid phase. For this reason, considerable effort has been devoted to determining the rate-limiting steps for transport of FA across pure lipid bilayer membranes. Previously, we found that transbilayer flip-flop was the rate-limiting step for transport of long chain anthroyloxy FA (AOFA) across lipid bilayers and that the times for long chain AOFA flip-flop were > or = 100 s, yielding rate constants for flip-flop (k(ff)) that were < or = 0.01 s(-1) [Storch, J., & Kleinfeld, A. M. (1986) Biochemistry 25, 1717-1726; Kleinfeld, A. M., & Storch, J. (1993) Biochemistry 32, 2053-2061]. In those studies, k(ff) values were inferred from the time course of AOFA transfer between lipid vesicles. Recently, Kamp et al. [Kamp, F., Zakim, D., Zhang, F., Noy, N., & Hamilton, J. A. (1995) Biochemistry 34, 11928-11937], using pyranine trapped within lipid vesicles to detect flip-flop more directly, have reported that flip-flop rates of long chain AOFA are extremely rapid (k(ff) > 10 s(-1)) and are not rate limiting for transbilayer transport. Because no defect was apparent in our previous measurements, we have extended, for AOFA, the pyranine method of Kamp et al. (1995) by using stopped-flow fluorometry to resolve flip-flop rates of both short and long chain AOFA in vesicles. In addition, we have monitored the time course of transbilayer AOFA flip-flop using carboxyfluorescein (CF) trapped within the lipid vesicles as a resonance energy transfer (RET) acceptor of AO fluorescence. The differential quenching of AOFA fluorescence in the outer and inner leaflets of the bilayer allows flip-flop to be separated from the time course of AOFA binding to the vesicles. Results obtained from both the pyranine and CF methods indicate, in agreement with our previous results, that flip-flop of the long chain AOFA is slow relative to either the binding or the rate of dissociation from the vesicle. In particular, we find that the time constant (tau) for pyranine quenching by 2-AO-palmitate (2-AOPA) was > 40 s and that k(ff) obtained from RET in CF vesicles was about 0.003 s(-1). Also, in contrast to Kamp et al. (1995) who reported that k(ff) values were independent of FA chain length or structure for the C-12 to C-18 native and the C-18 AOFA within a factor of 2, we find that the rate of pyranine quenching for the shorter chain 11-AO-undecanoic acid is more than 50-fold faster than for the longer chain AOFA. We conclude, therefore, that transbilayer transport of the AOFA is limited by the rate of flip-flop and that this rate is a sensitive function of the AOFA structure.

Fatty Acid Transfer from Liver and Intestinal Fatty Acid-binding Proteins to Membranes Occurs by Different Mechanisms

Intestinal absorptive cells contain high levels of expression of two homologous fatty acid-binding proteins (FABP), liver FABP (L-FABP), and intestinal FABP (I-FABP). Both bind long chain fatty acids with relatively high affinity. The functional distinction, if any, between these two proteins remains unknown. It is often hypothesized that FABP are important in intracellular transport of fatty acids. To assess whether fatty acid transport properties might differ between the two enterocyte FABPs, we examined the rate and mechanism of transfer of fluorescent anthroyloxy fatty acids (AOFA) from these proteins to model membranes using a resonance energy transfer assay. The results show that the absolute rate of AOFA transfer from I-FABP is faster than from L-FABP. Moreover, the apparent mechanism of fatty acid transfer is different between the two proteins. The rate of AOFA transfer from I-FABP is independent of ionic strength, directly dependent on the concentration of acceptor membrane vesicles, and dramatically regulated by the lipid composition of the membranes. These data strongly suggest that fatty acid transfer from I-FABP to membranes occurs by direct collisional interaction of the protein with the phospholipid bilayer. In contrast, the characteristics of fatty acid transfer from L-FABP are consistent with an aqueous diffusion-mediated process. Thus the two enterocyte FABPs may perform different functions within the intestinal absorptive cell in the regulation of fatty acid transport and utilization. It is hypothesized that L-FABP may act as a cytosolic buffer for fatty acids, maintaining the unbound fatty acid concentration, whereas I-FABP may be involved in the uptake and/or specific targeting of fatty acid to subcellular membrane sites.

The influence of an electric field on ion and water accessibility to stratum corneum lipid lamellae.

To study ion transport through stratum corneum (SC) lipid lamellae under passive and iontophoretic conditions. Iodide ion transport was measured by fluorescence quenching. Since the process involves diffusion of an iodide ion to the fluorophore located within the SC lamellae, the accessibility of iodide ions was measured. Moreover, the use of anthroyloxy fatty acid probes, provided information as a function of depth within the lamellae. Fluorescence quenching by iodide ions increased with iontophoretic current density, suggesting increased ion accessibility within the SC lamellae. In addition, at constant current, quenching decreased as the fluorophore was located deeper within the lamellae. This gradient in ion accessibility suggests that more iodide is found near the head-group than near the core of the SC lipid lamellae. Results obtained in the absence of iodide also show increased water accessibility during iontophoresis. These results show that in the presence of an applied electric field the SC lipid lamellae interior becomes more accessible to water and ions. These results imply that during iontophoresis, ion and water transport through human skin is associated, at least in part, with the SC lipid lamellae.

Molecular architecture and biophysical properties of phospholipids during thermal adaptation in fish: An experimental and model study

Phospholipids from livers of carps (Cyprinus carpio L.) adapted to winter (5 degrees C) and summer (25 degrees C) temperatures were isolated, and the fatty acid composition of total phospholipids, as well as molecular species composition of diacyl phosphatidylcholines and ethanolamines, were determined. Order parameter of 5-doxyl stearic acid and steady-state fluorescence anisotropy of different anthroyloxy fatty acids--[2-, 12(N-9-anthroyloxy)stearic acid and 16(N-9-anthroyloxy)palmitic acid--embedded in native and synthetic (16:0/16:0, 16:0/22:6, 18:0/22:6, 18:1/22:6, 20:4/20:4, 22:6/22:6 phosphatidylcholines and 16:0/18:1, 18:1/22:6 phosphatidylethanolamines) phospholipid vesicles was also determined between -30 and 30 degrees C and 5 and 30 degrees C, respectively. There is an accumulation of 1-monoenoic, 2-polyenoic diacyl phosphatidylcholine and ethanolamine with a concomitant reduction of 1-stearoyl,2-docosahexaenoyl species in the cold-adapted state. Despite a 30% accumulation of long-chain polyunsaturated fatty acids in phospholipids in cold, there is only a 5 degrees C downshift in the solid-gel to liquid-crystalline phase transition temperature (-8 vs. -13 degrees C). Vesicles from total phospholipids of cold-adapted fish proved to be more disordered in all segments than from the warm-adapted ones when assayed using 2,12-(N-9-anthroyloxy)stearic and 16-(N-9-anthroyloxy)palmitic acid. Vesicles made from purified phosphatidylcholines showed the same pattern, but they were more disordered than the corresponding total phospholipids. This could be modelled using mixed phospholipid vesicles made of synthetic 16:0/22:6 phosphatidylcholine (75%) and either 18:1/22:6 phosphatidylethanolamine (25%) vs. 16:0/18:1 phosphatidylethanolamine (25%) and comparison of the anisotropy parameters of 100% 16:0/22:6 and 100% 18:1/22:6 phosphatidylcholine vesicles. Mixing either 16:0/18:1 (25%) or 18:1/22:6 (25%) phosphatidylethanolamines to 18:0/22:6 (75%) phosphatidylcholine shifted down or up, respectively, the transition temperature of vesicles compared to 100% 18:0/22:6 vesicles assayed by electron spin resonance spectroscopy using 5-doxylstearic acid. It is concluded that it is not the gross amount of long-chain polyunsaturated fatty acids in phospholipids, but rather their specific combination with cis delta 9 monounsaturated fatty acids in the position sn-1, especially in phosphatidylethanolamines, that is important in determining the physical properties of biomembranes in relation to adaptational temperature.

Fluorescence spectroscopy of 9-anthroyloxy fatty acids in solvents

A series of anthroyloxy fatty acid (AF) fluorescent probes, with the anthroyloxy group covalently linked at various positions along the alkyl chain, were studied in solvents exhibiting a wide range of polarity and hydrogen-bond donor (H d) and acceptor (H a) ability. These probes were sensitive to the solvent polarity as reflected by the Stokes' shift observed in steady state fluorescence. As determined by multi-linear regression analysis of the observed Stokes' shift and solvent parameters, such as orientation polarizability ( Δf), H d and H a of the solvents, all the probes were sensitive to the H d of solvents but were not affected by the H a of solvents except the 2-AF. Due to the proximity of the polar headgroup to the fluorophore, it appears that some intramolecular hydrogen-bonding is present in 2-AF, an interaction that is sensitive to the pH of the solvent, but is less sensitive to the H d and H a of the solvents. Fluorescence lifetimes measured by the multi-frequency phase-modulation technique in mixtures of hexane and ethanol reflect a modified Stern-Volmer behavior suggesting the second solvent, ethanol, specifically interacts with the probe, in part through collisional quenching. Also, the lifetime data were sensitive to very low concentrations of the second solvent (0–0.1%, by vol.). The results from this study provide insight into the intrinsic differences between the different AF positions that must be taken into consideration while investigating the dynamics of lipid bilayer systems. Moreover, this study illustrates the utility and resolving power of lifetime based measurements needed for the interpretation of heterogeneous biophysical environments.

Cell membrane microviscosity and Ca(2+)-Mg(2+)-ATPase activity do not contribute to hypertension in the spontaneously hypertensive rat model.

1. The relationships between systolic blood pressure and altered erythrocyte Ca(2+)-Mg(2+)-ATPase activity and membrane microviscosity were assessed in membranes prepared from 20-week-old female Wistar-Kyoto normotensive and spontaneously hypertensive rats obtained from two different sources (Charles River and Harlan OLAC) and a second filial (F2) generation derived from a cross between Wistar-Kyoto rats and spontaneously hypertensive rats from one source (Charles River). 2. Spontaneously hypertensive rats from both sources had systolic blood pressures significantly higher than those of Wistar-Kyoto animals (P < 0.05; 151 +/- 4 and 110 +/- 3 mmHg, Charles River; 155 +/- 4 and 122 +/- 4 mmHg, Harlan OLAC). The systolic blood pressures for the F2 rat population ranged between 73 and 168 mmHg. 3. Ca(2+)-Mg(2+)-ATPase activity was measured as ATP-dependent 45Ca2+ uptake into inside-out vesicles and microviscosity assessed by the measurement of polarization anisotropy of membrane incorporated fluorescent probes including 1,6-diphenyl-1,3,5-hexatriene, trimethylamino-1,6-diphenyl-1,3,5-hexatriene and a series of anthroyloxy fatty acids. 4. Contrary to previous studies, no relationship between adult systolic blood pressure and erythrocyte Ca(2+)-Mg(2+)-ATPase activity or general or localized membrane microviscosity was indicated by the comparison of spontaneously hypertensive and Wistar-Kyoto animals or in the analysis of the F2 rat population. 5. These results suggest that Ca(2+)-Mg(2+)-ATPase activity and membrane microviscosity are causally unrelated to hypertension in these animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Diversity of fatty acid-binding protein structure and function: studies with fluorescent ligands.

The mammalian fatty acid-binding proteins (FABP) are localized in many distinct cell types. They bind long chain fatty acids in vitro, however, their functions and mechanisms of action in vivo remain unknown. The present studies have sought to understand the relationships among these proteins, and to address the possible role of FABP in cellular fatty acid traffic. A series of anthroyloxy-labeled fluorescent fatty acids have been used to examine the physicochemical properties of the fatty acid-binding sites of different members of the FABP family. The fatty acid probes have also been used to study the rate and mechanism of fatty acid transfer from different FABP types to phospholipid membranes. The results of these studies show a number of interesting and potentially important differences between FABP family members. An examination of adipocyte and heart FABP (A- and H-FABP) shows that their fatty acid-binding sites are less hydrophobic than the liver FABP (L-FABP) site, and that the bound ligand experiences less motional constraint within the A- and H-FABP binding sites than within the L-FABP binding site. In keeping with these differences in structural properties, it was found that anthroyloxy-fatty acid transfer from A- and H-FABP to membranes is markedly faster than from L-FABP. Moreover, the mechanism of fatty acid transfer was found to be similar for the highly homologous A- and H-FABP, whereby transfer to phospholipid membranes appears to occur via transient collisional interactions between the FABP and membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of antioxidants with depth-dependent fluorescence quenchers and energy transfer probes in lipid bilayers

Three fluorescent, lipophilic, heterocyclic antioxidants were incorporated into lipid bilayers and exposed to depth-dependent nitroxyl fatty acid quenchers. The Stern-Volmer plots curved upward at low quencher concentrations. Quantitative analysis of the results showed that this behavior is consistent with complex formation between quencher and fluorescent antioxidant, where the complex is 2–3 times more fluorescent than the parent fluorophore. At higher quencher concentrations, both free antioxidant and ‘bright complex’ are quenched dynamically, albeit quenching of the latter is less efficient. The complex probably results from ionic, hydrogen bond and π-π interactions. Formation of such a ‘bright complex’ is also observable in a homogeneous solution of the reactants in cyclohexane. Additional evidence for the complexation of these antioxidants with fatty acids in lipid bilayers is provided by the fact that energy transfer from the antioxidants to anthroyloxy fatty acids occurs at surface concentrations where radiative energy transfer between free molecules should not be efficient. For directly probing the relative depths of these fluorophores in lipid bilayers we used the aqueous quenchers acrylamide and iodide. They showed that, in terms of increasing depth in the bilayer, the order was U-78,517f < U-78,518e < U-75,412e. Our results, in toto, demonstrate that the Lazaroid antioxidants are incorporated into the lipid bilayer where they occupy strictly defined positions and orientations. Complexation with fatty acyl chains should be mechanistically relevant, since it may enhance antioxidant activity by hindering free radical chain propagation.

Compression of lipid membranes as observed at varying membrane positions

We have measured the microscopic isothermal compressibility of dioleoyl- and dimyristyl-phosphatidylcholine multilayers and bilayers as a function of membrane depth by the pressure dependence of the polarization of a series of anthroyloxy fatty acids. In both systems, within experimental error, the compressibility did not change with membrane depth. The magnitudes of the compressibilities matched those of organic solids and those reported for dipalmitoylphosphatidylcholine multilayers from neutron diffraction measurements (Braganza, L. F., and D. L. Worcester. 1986. Biochemistry, 25:7484-7488). The bilayer compressibility decreased with temperature and this decrease was similar with membrane depth consistent with the isotropic thermal expansion of membranes previously observed (Scarlata, S. 1989. Biophys. J. 55:1215-1223). The vertical compressibility in the z direction is much lower than the horizontal (xy planes) for probes that lie parallel to the hydrocarbon chains which is consistent with an increase in bilayer thickness. The compressibility for probes that lie perpendicular to the hydrocarbon chains is more isotropic due to their limited spatial access to the z plane.

Evaluation of the thermal coefficient of the resistance to fluorophore rotation in model membranes

The thermal coefficient of the frictional resistance to fluorophore rotation (b), a parameter related to the change in the local viscosity with temperature, was determined for anthroyloxy-fatty acid probes in micelles and dimyristoyl lecithin (DMPC) and dioleoyl lecithin (DOPC) unilamellar and multilamellar vesicles. The value of b and the percent change in anisotropy with temperature (%dA/dT) remained constant with membrane depth and only depended on composition. These parameters were also the same when either in-plane, or in-plane and out-of-plane fluorophore motions were observed. This result indicates that the membranes expand isotropically. The magnitude of b was found to be primarily dependent on the packing of the hydrocarbon chains with higher b values relating to more closely-packed chains. b was responsive to the gel to liquid crystal phase transition of DMPC and the bilayer to hexagonal phase transition of egg-phosphatidylethanolamine. When the enthalpy values for the fluorophore transfer from one phase to another are calculated, the values are larger than those measured by calorimetry and reflect a discrepancy between the microscopic enthalpy experienced by the fluorophore due to a change in environment versus the macroscopic enthalpy of the system as a whole.

Fluorescence depolarization measurements on oriented membranes

We describe the theory and experimental application of fluorescence depolarization measurements on small molecules bound to oriented phospholipid bilayers. The results yield insight into both the orientation and the rotational motion of fluorophores in a membrane environment. To accomplish this the angular distribution of polarized fluorescence intensities is measured on a membrane preparation consisting of stacked phospholipid bilayers oriented in a known coordinate system. Considerably more information is available from this data than in comparable solution phase measurements. Three parameters are derived from the data: the rate of rotational diffusion and the second and fourth degree order parameters. These latter two parameters provide an assessment of the average distribution of fluorophore orientation in the membrane bilayer. The data have been carefully examined for systematic experimental artifacts and new protocols are presented which help to eliminate errors that have not been amply treated in the past. We present data for two types of fluorescent molecules: (a) conventional membrane probes like diphenylhexatriene, perylene and anthroyloxy fatty acids; and (b) the anticancer agent adriamycin and several congeneric anthracycline antibiotics. The results show that the hydrocarbon core of membranes is more rigid than previously thought, particularly above the thermal phase transition temperature. We also show that the orientation of small molecules is sensitive to both the phospholipid composition and to the interaction of specific functional groups with the lipid bilayer. The results are discussed in terms of energetic models describing the general patterns for the binding of small molecules to biological membranes.

Interactions of phenytoin with rat brain synaptosomes examined by fluorescent fatty acid probes

Phenytoin (PHT) modified the fluorescent characteristics of anthroyloxy-fatty acids in synaptosomal membranes. Association of PHT with synaptosomal membranes caused the greatest change when the fluorescent probe was located at the 6-carbon position of N-(anthroyloxy)stearic acid and was incorporated into the membranes. Phenytoin and 6-(anthroyloxy)stearic acid compete for high affinity binding regions which are probably lipid domains within the membrane. Phenytoin has a weaker association with the sites than the fluorescent fatty acids. Divalent cations, e.g. Mg(2+) or Ca(2+), are required to observe maximal change in polarization of fluorescence of fatty acid probes in the presence of PHT. It is proposed that the membrane lipid bilayer reorganizes to accommodate exogenous compounds, such as phenytoin or the fatty acid probe in order to permit the most efficient packing of lipids. This reorganization of the lipid bilayer may influence membrane enzyme activities and ion channels.