Diffusion of hydrogen peroxide across DPPC large unilamellar liposomes

The decomposition of hydrogen peroxide catalyzed by catalase entrapped in the pool of di-palmitoylphosphatidyl choline unilamellar liposomes has been studied. The rate of the process was evaluated by following the production of oxygen as a function of time. Under the experimental conditions employed the rate of oxygen production was controlled by the diffusion of hydrogen peroxide, allowing for the estimation of the diffusion coefficient of hydrogen peroxide across the liposome bilayer. The rate of diffusion across the bilayer increases with the temperature and the presence of fluidizers (n-nonanol), according with changes in the bilayer fluidity, as sensed by 1,6-diphenyl hexatriene (DPH) fluorescence anisotropy. A peculiar aspect of the data is the fast hydrogen peroxide diffusion observed at the bilayer phase transition temperature. This fast diffusion is associated to rafts fluctuations that take place in the partially melted bilayer. These fluctuations have no effect on the microviscosity sensed by DPH.     

Diffusion measurements of water, ubiquinone and lipid bilayer inside a cylindrical nanoporous support: a stimulated echo pulsed-field gradient MAS-NMR investigation

Stimulated echo pulsed-field gradient 1H magic angle spinning NMR has been used to investigate the mobility of water, ubiquinone and tethered phospholipids, components of a biomimetic model membrane. The diffusion constant of water corresponds to an isotropic motion in a cylinder. When the lipid bilayer is obtained after the fusion of small unilamellar vesicles, the extracted value of lipid diffusion indicates unrestricted motion. The cylindrical arrangement of the lipids permits a simplification of data analysis since the normal bilayer is perpendicular to the gradient axis. This feature leads to a linear relation between the logarithm of the attenuation of the signal intensity and a factor depending on the gradient strength, for lipids covering the inner wall of aluminium oxide nanopores as well as for lipids adsorbed on a polymer sheet rolled into a cylinder. The effect of the bilayer formation on water diffusion has also been observed. The lateral diffusion coefficient of ubiquinone is in the same order of magnitude as the lipid lateral diffusion coefficient, in agreement with its localization within the bilayer.     

Diffusion measurements of water, ubiquinone and lipid bilayer inside a cylindrical nanoporous support: A stimulated echo pulsed-field gradient MAS-NMR investigation

Stimulated echo pulsed-field gradient ¹H magic angle spinning NMR has been used to investigate the mobility of water, ubiquinone and tethered phospholipids, components of a biomimetic model membrane. The diffusion constant of water corresponds to an isotropic motion in a cylinder. When the lipid bilayer is obtained after the fusion of small unilamellar vesicles, the extracted value of lipid diffusion indicates unrestricted motion. The cylindrical arrangement of the lipids permits a simplification of data analysis since the normal bilayer is perpendicular to the gradient axis. This feature leads to a linear relation between the logarithm of the attenuation of the signal intensity and a factor depending on the gradient strength, for lipids covering the inner wall of aluminium oxide nanopores as well as for lipids adsorbed on a polymer sheet rolled into a cylinder. The effect of the bilayer formation on water diffusion has also been observed. The lateral diffusion coefficient of ubiquinone is in the same order of magnitude as the lipid lateral diffusion coefficient, in agreement with its localization within the bilayer.     

Pressure variation of the lateral diffusion in lipid bilayer membranes

A pressure-induced decrease of the lateral diffusion in pure and cholesterol containing phosphatidylcholine bilayer membranes has been determined by the excimer formation technique using pyrene as probe molecule. The experimental results at pressures up to 150 bars are described satisfactorily by the free volume theory of a molecular transport in liquids. A pressure increase of extrapolated 575 bars decreases the lateral diffusion of lipids by a factor of two in pure dipalmitoylphosphatidylcholine membranes. Higher pressures are necessary to induce the same effect in cholesterol containing membranes. This result is interpreted by the condensing effect of cholesterol in fluid bilayer membranes.     

The rotational diffusion of cytochrome b5 in lipid bilayer membranes. Influence of the lipid physical state.

A derivative of the integral membranes protein, cytochrome b5, has been prepared in which the native heme group has been replaced by the structurally similar rhodium(III)-protoporphyrin IX. This metalloporphyrin has a finite triplet yield with a single exponential decay time of 22 microsecond in water. After insertion of the metalloporphyrin into the protein, its triplet-state decay becomes strongly nonexponential with at least three equal amplitude components with time constants varying over a range of 100. The derivatized protein has been incorporated into unilamellar liposomes prepared from dimyristoyllecithin, and the rotational diffusion of the protein in the lipid bilayer has been studied at temperatures above and below the lipid phase transition temperature via triplet absorbance anisotropy decay. The anisotropy decay curves are biphasic both above and below the lipid phase transition. The rotational diffusion constant is found to be 2.4 X 10(5) s-1 at 35 degrees C, and 1.1 X 10(4) s-1 at 10 degrees C, both being calculated from the fast decay component. The ratio of the limiting anisotropy to the initial anisotropy is 0.6 at both temperatures. This implies a cone of restricted motion of 34 degrees for the protein in the bilayer.     

Diffusion of dihydropyridine calcium channel antagonists in cardiac sarcolemmal lipid multibilayers.

A membrane bilayer pathway model has been proposed for the interaction of dihydropyridine (DHP) calcium channel antagonists with receptors in cardiac sarcolemma (Rhodes, D.G., J.G. Sarmiento, and L.G. Herbette. 1985. Mol. Pharmacol. 27:612-623) involving drug partition into the bilayer with subsequent receptor binding mediated (though probably not rate-limited) by diffusion within the bilayer. Recently, we have characterized the partition step, demonstrating that DHPs reside, on a time-average basis, near the bilayer hydrocarbon core/water interface. Drug distribution about this interface may define a plane of local concentration for lateral diffusion within the membrane. The studies presented herein examine the diffusional dynamics of an active rhodamine-labeled DHP and a fluorescent phospholipid analogue (DiIC16) in pure cardiac sarcolemmal lipid multibilayer preparations as a function of bilayer hydration. At maximal bilayer hydration, the drug diffuses over macroscopic distances within the bilayer at a rate identical to that of DiI (D = 3.8 X 10(-8) cm2/s), demonstrating the overall feasibility of the membrane diffusion model. The diffusion coefficients for both drug and lipid decreased substantially as the bilayers were dehydrated. While identical at maximal hydration, drug diffusion was significantly slower than that of DiIC16 in partially dehydrated bilayers, probably reflecting differences in mass distribution of these probes in the bilayer.     

Molecular study of the diffusional process of DMSO in double lipid bilayers

As a way to quantify the diffusion process of molecular compounds through biological membranes, we investigated in this study the dynamics of DMSO through an 1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC) bilayer system. To properly account for the diffusion of DMSO due to a concentration gradient, a double DPPC bilayer was setup for our simulations. In such configuration, the aqueous phases can be explicitly associated with the extra and intracellular domains of the membrane, which is seldom the case in studies of single lipid bilayer due to the periodicity imposed by the simulations. DMSO molecules were initially contained in one of the aqueous phases (extracellular region) at a concentration of 5 wt.%. Molecular dynamics simulation was performed in this system for 95 ns at 350 K and 1 bar. The simulations showed that although many DMSO molecules penetrated the lipid bilayer, only about 10% of them crossed the bilayer to reach the other aqueous phase corresponding to the intracellular region of the membrane. The simulation time considered was insufficient to reach equilibrium of the DMSO concentration between the aqueous phases. However, the simulations provided sufficient information to estimate parameters to apply Fick's Law to model the diffusion process of the system. Using this model, we predicted that for the time considered in our simulation, the concentration of DMSO in the intracellular domain should have been about half of the actual value obtained. The model also predicted that equilibrium of the DMSO concentration in the system would be reached after about 2000 ns, approximately 20 times longer than the performed simulation.     

Diffusional dynamics of an active rhodamine-labeled 1,4-dihydropyridine in sarcolemmal lipid multibilayers.

A "membrane bilayer pathway" model, involving ligand partition into the bilayer, lateral diffusion, and receptor binding has been invoked to describe the 1,4-dihydropyridine (DHP) calcium channel antagonist receptor binding mechanism. In an earlier study (Chester et al. 1987. Biophys. J. 52:1021-1030), the diffusional component of this model was examined using an active fluorescence labeled DHP calcium channel antagonist, nisoldipine-lissamine rhodamine B (Ns-R), in purified cardiac sarcolemmal (CSL) lipid multibilayers. Diffusion coefficient measurements on membrane-bound drug and phospholipid at maximum bilayer hydration yielded similar values (3.8 x 10(-8) cm2/s). However, decreases in bilayer hydration resulted in dramatically reduced diffusion coefficient values for both probes with substantially greater impact on Ns-R diffusion. These data suggested that hydration dependent diffusional differences could be a function of relative probe location along the bilayer normal. In this communication, we have addressed the relative effect of the rhodamine substituent on Ns-R diffusion complex by examining the diffusional dynamics of free rhodamine B under the same conditions used to evaluate Ns-R complex and phospholipid diffusion. X-ray diffraction studies were performed to determine the Ns-R location in the membrane and model the CSL lipid bilayer profile structure to give a rationale for the differences in probe diffusional dynamics as a function of interbilayer water space.     

Electrochemical measurement of lateral diffusion coefficients of ubiquinones and plastoquinones of various isoprenoid chain lengths incorporated in model bilayers.

The long-range diffusion coefficients of isoprenoid quinones in a model of lipid bilayer were determined by a method avoiding fluorescent probe labeling of the molecules. The quinone electron carriers were incorporated in supported dimyristoylphosphatidylcholine layers at physiological molar fractions (<3 mol%). The elaborate bilayer template contained a built-in gold electrode at which the redox molecules solubilized in the bilayer were reduced or oxidized. The lateral diffusion coefficient of a natural quinone like UQ10 or PQ9 was 2.0 +/- 0.4 x 10(-8) cm2 s(-1) at 30 degrees C, two to three times smaller than the diffusion coefficient of a lipid analog in the same artificial bilayer. The lateral mobilities of the oxidized or reduced forms could be determined separately and were found to be identical in the 4-13 pH range. For a series of isoprenoid quinones, UQ2 or PQ2 to UQ10, the diffusion coefficient exhibited a marked dependence on the length of the isoprenoid chain. The data fit very well the quantitative behavior predicted by a continuum fluid model in which the isoprenoid chains are taken as rigid particles moving in the less viscous part of the bilayer and rubbing against the more viscous layers of lipid heads. The present study supports the concept of a homogeneous pool of quinone located in the less viscous region of the bilayer.     

Molecular study of the diffusional process of DMSO in double lipid bilayers

As a way to quantify the diffusion process of molecular compounds through biological membranes, we investigated in this study the dynamics of DMSO through an 1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC) bilayer system. To properly account for the diffusion of DMSO due to a concentration gradient, a double DPPC bilayer was setup for our simulations. In such configuration, the aqueous phases can be explicitly associated with the extra and intracellular domains of the membrane, which is seldom the case in studies of single lipid bilayer due to the periodicity imposed by the simulations. DMSO molecules were initially contained in one of the aqueous phases (extracellular region) at a concentration of 5 wt.%. Molecular dynamics simulation was performed in this system for 95 ns at 350 K and 1 bar. The simulations showed that although many DMSO molecules penetrated the lipid bilayer, only about 10% of them crossed the bilayer to reach the other aqueous phase corresponding to the intracellular region of the membrane. The simulation time considered was insufficient to reach equilibrium of the DMSO concentration between the aqueous phases. However, the simulations provided sufficient information to estimate parameters to apply Fick's Law to model the diffusion process of the system. Using this model, we predicted that for the time considered in our simulation, the concentration of DMSO in the intracellular domain should have been about half of the actual value obtained. The model also predicted that equilibrium of the DMSO concentration in the system would be reached after about 2000 ns, approximately 20 times longer than the performed simulation.     

An NMR study of the temperature dependence of the coefficient of water self-diffusion through lipid bilayer membranes

The temperature dependence of the coefficient of water self-diffusion through plane-parallel lipid multilayers of the phospholipid dioleoylphosphatidylcholine oriented on a glass support has been studied in the temperature range of 20-60 degrees C by the method of NMR with magnetic field pulse gradient. The values of the coefficients of transbilayer water diffusion are by four orders of magnitude less than for bulky water and ten times less than the coefficients of lateral diffusion of the lipid under the same conditions. The temperature dependence of the coefficient of water diffusion is described by the Arrhenius law with an apparent activation energy of about 41 kJ/mol, which far exceeds the activation energy for the diffusion of bulky water (18 kJ/mol). The experimental data were analyzed using a "dissolving-diffusion" model, by simulating the passage of water through membrane channels, and by analyzing the exchange of water molecules in states with different modes of translation mobility, including pore channels and bilayer "defects". Each of the approaches used made it possible to take the significance of bilayer permeability for the apparent energy of activation of water diffusion into account and estimate the energies of activation of water diffusion in the hydrophobic moiety of the bilayer, which were found to be close to the values for bulky water. The coefficients of water diffusion in the system under examination and the coefficients of permeation of water through the bilayer were estimated, and the effect of bilayer "defects" on the coefficients of water diffusion along and across bilayers was studied.     

Localization and preferred orientations of ubiquinone homologs in model bilayers.

The localization of ubiquinone has been investigated in phospholipid bilayer vesicles in studies of fluorescence quenching of membrane-bound probes by ubiquinone homologs (Qn, where n is the number of the isoprenoid units of the chain). Fluorescence-quenching data obtained by using a set of anthroylstearate probes, having the fluorophore located at different depths, revealed that ubiquinone-3 is located throughout the whole bilayer thickness. From the bimolecular quenching constants in the membrane, lateral diffusion coefficients in two dimensions were calculated to span values of 10(-7)-10(-6) cm2.s-1. This suggests that ubiquinones laterally diffuse in a very fluid environment. On this basis, it is proposed that their translational diffusion in the bilayer takes place in two dimensions, with the quinone ring oscillating between the two bilayer surfaces within a hydrophobic environment not extending beyond the glycerol region. This model implies that the quinonic head is both settled near the polar surface of the bilayer and buried into the host hydrocarbon interior. This two-site distribution was confirmed for all Qn, except Q0, by their linear dichroism spectra in the bilayers provided by disc-like lyotropic nematic liquid crystals. These spectra also provided detailed information on the preferential orientations of the quinonic head of the different derivatives within the two sites. The mechanism by which the localization and orientation of Qn guest molecules inside the host bilayer is modulated by the isoprenoid chain length is discussed on a thermodynamical basis. Being that Qn is expected to be also widely contained in the highly curved cristae of the mitochondrial inner membrane, by using rod-like lyotropic nematic liquid crystals we searched out effects of the curvature of the host bilayer on those Qn distributions. The linear dichroism measurements reveal that Qn guest molecules are no longer obliged to find a partition between two different types of localizations when the host bilayer is highly curved. In this case all Qn, even the longest Q10, were found to stay parallel to the amphiphilic chains with a single site localization of the head near the polar interface. By the same linear dichroism technique, the local ordering of all Qn derivatives was also evaluated. The order parameters were found to be basically the same for all derivatives. This result is justified on the basis of the relaxation, caused by the surface curvature, of the lateral compression of the host chains.     

Transport methods for probing the barrier domain of lipid bilayer membranes.

Two experimental techniques have been utilized to explore the barrier properties of lecithin/decane bilayer membranes with the aim of determining the contributions of various domains within the bilayer to the overall barrier. The thickness of lecithin/decane bilayers was systematically varied by modulating the chemical potential of decane in the annulus surrounding the bilayer using different mole fractions of squalene in decane. The dependence of permeability of a model permeant (acetamide) on the thickness of the solvent-filled region of the bilayer was assessed in these bilayers to determine the contribution of this region to the overall barrier. The flux of acetamide was found to vary linearly with bilayer area with Pm = (2.9 +/- 0.3) x 10(-4) cm s-1, after correcting for diffusion through unstirred water layers. The ratio between the overall membrane permeability coefficient and that calculated for diffusion through the hydrocarbon core in membranes having maximum thickness was 0.24, suggesting that the solvent domain contributes only slightly to the overall barrier properties. Consistent with these results, the permeability of acetamide was found to be independent of bilayer thickness. The relative contributions of the bilayer interface and ordered hydrocarbon regions to the transport barrier may be evaluated qualitatively by exploring the effective chemical nature of the barrier microenvironment. This may be probed by comparing functional group contributions to transport with those obtained for partitioning between water and various model bulk solvents ranging in polarity or hydrogen-bonding potential. A novel approach is described for obtaining group contributions to transport using ionizable permeants and pH adjustment. Using this approach, bilayer permeability coefficients of p-toluic acid and p-hydroxymethyl benzoic acid were determined to be 1.1 +/- 0.2 cm s-1 and (1.6 +/- 0.4) x 10(-3) cm s-1, respectively. From these values, the -OH group contribution to bilayer transport [delta(delta G0-OH)] was found to be 3.9 kcal/mol. This result suggests that the barrier region of the bilayer does not resemble the hydrogen-bonding environment found in octanol, but is somewhat less selective (more polar) than a hydrocarbon solvent.     

DNA-containing liposomes as a model for the study of cell membrane permeation by anthracycline derivatives

The uptake of anthracycline derivatives into large unilamellar vesicles (LUV) in response to a driven force provided by DNA encapsulated inside the LUV has been investigated. Four anthracyclines have been used: adriamycin, 4'-O-tetrahydropyranyladriamycin (THP-ADR), daunorubicin (DNR), and carminomycin. No quenching of the drug fluorescence is observed through interaction of the drugs with the lipidic bilayer. Rapid quenching of drug fluorescence occurs when drugs intercalate between the base pairs of DNA. The kinetics of the decay of anthracycline fluorescence in the presence of DNA-containing liposomes can thus be used to follow the diffusion of the drug through the membrane. The initial rates of uptake, as a function of pH, and lipid bilayer permeability coefficients have been calculated for the neutral forms of THP-ADR and DNR. This system suggests that anthracycline may gain access to cells by passive diffusion of the neutral form of the drug under the action of a driven force provided by DNA in the nucleus.     

The polymer-supported phospholipid bilayer: tethering as a new approach to substrate-membrane stabilization

We present a new molecular engineering approach in which a polymer-supported phospholipid bilayer is vertically stabilized by controlled covalent tethering at both the polymer-substrate and polymer-bilayer interfaces. This approach is based on lipopolymer molecules, which not only form a polymer cushion between the phospholipid bilayer and a solid glass substrate but also act as covalent connections (tethers) between the bilayer and cushion. Our approach involves Langmuir-Blodgett transfer of a phospholipid-lipopolymer monolayer followed by Schaefer transfer of a pure phospholipid monolayer and is capable of varying the tethering density between the polymer layer and the phospholipid bilayer in a very controlled manner. Further stabilization is achieved if the glass substrate is surface-functionalized with a benzophenone silane. In this case, a photocross-linking reaction between the polymer and benzophenone group allows for the covalent attachment of the polymer cushion to the glass substrate. This approach is similar to that recently reported by Wagner and Tamm in which double tethering is achieved via lipopolymer silanes (Wagner, M. L.; Tamm, L. K. Biophys. J. 2000, 79, 1400). To obtain a deeper understanding of how the covalent tethering affects the lateral mobility of the bilayer, we performed fluorescence recovery after photobleaching (FRAP) experiments on polymer-tethered bilayers at different tethering densities (lipopolymer/phospholipid molar ratios). The FRAP data clearly indicate that the hydrophobic lipopolymer moieties act as rather immobile obstacles within the phospholipid bilayer, thereby leading to hindered diffusion of phospholipids. Whereas the high lateral diffusion coefficient of D = 17.7 mum(2)/s measured at low tethering density (5 mol % lipopolymer) indicates rather unrestricted motion within the bilayer, corresponding values at moderate (10 mol % lipopolymer) and high (30 mol % lipopolymer) tethering densities of D = 9.7 mum(2)/s and D = 1.1 mum(2)/s, respectively, show significant hindered diffusion. These results are contrary to the recent findings on similar membrane systems reported by Wagner and Tamm in which no significant change in phospholipid diffusion was found between 0 and 10 mol % lipopolymer. Our experimental report leads to a deeper understanding of the complex problem of interlayer coupling and offers a path toward a compromise between stability of the whole system and lateral mobility within the bilayer. Furthermore, the FRAP measurements show that polymer-tethered membranes are very interesting model systems for studying problems of restricted diffusion within two-dimensional fluids.     

Mesoscopic simulation of cell membrane damage, morphology change and rupture by nonionic surfactants.

A new simulation method, dissipative particle dynamics, is applied to model biological membranes. In this method, several atoms are united into a single simulation particle. The solubility and compressibility of the various liquid components are reproduced by the simulation model. When applied to a bilayer of phosphatidylethanolamine, the membrane structure obtained matches quantitatively with full atomistic simulations and with experiments reported in the literature. The method is applied to investigate the cause of cell death when bacteria are exposed to nonionic surfactants. Mixed bilayers of lipid and nonionic surfactant were studied, and the diffusion of water through the bilayer was monitored. Small transient holes are seen to appear at 40% mole-fraction C(9)E(8), which become permanent holes between 60 and 70% surfactant. When C(12)E(6) is applied, permanent holes only arise at 90% mole-fraction surfactant. Some simulations have been carried out to determine the rupture properties of mixed bilayers of phosphatidylethanolamine and C(12)E(6). These simulations indicate that the area of a pure lipid bilayer can be increased by a factor 2. The inclusion of surfactant considerably reduces both the extensibility and the maximum stress that the bilayer can withstand. This may explain why dividing cells are more at risk than static cells.     

Determination of partition and lateral diffusion coefficients of ubiquinones by fluorescence quenching of n-(9-anthroyloxy)stearic acids in phospholipid vesicles and mitochondrial membranes

The quenching of fluorescence of n-(9-anthroyloxy)stearic acids and other probes by different ubiquinone homologues and analogues has been exploited to assess the localization and lateral mobility of the quinones in lipid bilayers of model and mitochondrial membranes. The true bimolecular collisional quenching constants in the lipids together with the lipid/water partition coefficients were obtained from Stern-Volmer plots at different membrane concentrations. A monomeric localization of the quinone in the phospholipid bilayer is suggested for the short side-chain ubiquinone homologues and for the longer derivatives when cosonicated with the phospholipids. The diffusion coefficients of the ubiquinones, calculated from the quenching constants either in three dimensions or in two dimensions, are in the range of (1-6) X 10(-6) cm2 s-1, both in phospholipid vesicles and in mitochondrial membranes. A careful analysis of different possible locations of ubiquinones in the phospholipid bilayer, accounting for the calculated diffusion coefficients and the viscosities derived therefrom, strongly suggests that the ubiquinone 10 molecule is located within the lipid bilayer with the quinone ring preferentially adjacent to the polar head groups of the phospholipids and the hydrophobic tail largely accommodated in the bilayer midplane. The steady-state rates of either ubiquinol 1-cytochrome c reductase or NADH:ubiquinone 1 reductase are proportional to the concentration of the quinol or quinone substrate in the membrane. The second-order rate constants appear to be at least 3 orders of magnitude lower than the second-order constants for quenching of the fluorescent probes; this is taken as a clear indication that ubiquinone diffusion is not the rate-determining step in the quinone-enzyme interaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anisotropic motion of cholesterol in oriented DPPC bilayers studied by quasielastic neutron scattering: the liquid-ordered phase.

Quasielastic neutron scattering (QENS) at two energy resolutions (1 and 14 microeV) was employed to study high-frequency cholesterol motion in the liquid ordered phase (lo-phase) of oriented multilayers of dipalmitoylphosphatidylcholine at three temperatures: T = 20 degrees C, T = 36 degrees C, and T = 50 degrees C. We studied two orientations of the bilayer stack with respect to the incident neutron beam. This and the two energy resolutions for each orientation allowed us to determine the cholesterol dynamics parallel to the normal of the membrane stack and in the plane of the membrane separately at two different time scales in the GHz range. We find a surprisingly high, model-independent motional anisotropy of cholesterol within the bilayer. The data analysis using explicit models of molecular motion suggests a superposition of two motions of cholesterol: an out-of-plane diffusion of the molecule parallel to the bilayer normal combined with a locally confined motion within the bilayer plane. The rather high amplitude of the out-of-plane diffusion observed at higher temperatures (T >/= 36 degrees C) strongly suggests that cholesterol can move between the opposite leaflets of the bilayer while it remains predominantly confined within its host monolayer at lower temperatures (T = 20 degrees C). The locally confined in-plane cholesterol motion is dominated by discrete, large-angle rotational jumps of the steroid body rather than a quasicontinous rotational diffusion by small angle jumps. We observe a significant increase of the rotational jump rate between T = 20 degrees C and T = 36 degrees C, whereas a further temperature increase to T = 50 degrees C leaves this rate essentially unchanged.     

Transport of fatty acids across membranes by the diffusion mechanism

In early research on fatty acid transport, passive diffusion seemed to provide an adequate explanation for movement of fatty acids through the membrane bilayer. This simple hypothesis was later challenged by the discovery of several proteins that appeared to be membrane-related fatty acid transporters. In addition, some biophysical studies suggested that fatty acids moved slowly through the simple model membranes (phospholipid bilayers), which would provide a rationale for protein-assisted transport. Furthermore, it was difficult to rationalize how fatty acids could diffuse passively across the bilayer as anions. Newer studies have shown that fatty acids are present in membranes in the un-ionized as well as the ionized form, and that the un-ionized form can cross a protein-free phospholipid bilayer quickly. This flip-flop mechanism has been validated in cells by intracellular pH measurements. The role of putative fatty acid transport proteins remains to be clarified.