An ESR characterization of micelles and vesicles formed in aqueous decanoic acid/sodium decanoate systems using different spin labels

Aqueous decanoic acid/sodium decanaote systems were studied as a function of pH and concentration, up to 0.3 M decanoic acid/sodium decanoate, by electron spin resonance (ESR) spectroscopy using three different amphiphilic spin labels. The distribution of the spin labels between vesicles and micelles as well as their dynamic properties were determined by quantitative analysis of the ESR spectra using two novel simulation software packages. Rotational correlation time of the labels in micelles was found to increase with decreasing pH, with substantial increase in the region where vesicles were formed (7.8相似文献   

Matrix Effect in Oleate Micelles-Vesicles Transformation

It is accepted by many authors that the formation of closed molecular structures is a key step in the evolution of life. Oleate vesicles represent a good model system in this framework due to the fact that they self-assemble spontaneously and that fatty acids are considered as possible prebiotic structures. In this contribution, we will focus the attention on the transition from oleate micelles to oleic acid/oleate vesicles induced by a pH change. This transformation is strongly influenced by the presence of pre-formed vesicles. We called this phenomenon the matrix effect. The influence of pre-added POPC liposomes (POPC = 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine) and oleic acid/oleate vesicles on the process rate and on the final size distribution will be discussed elucidating the main differences between these two systems.     

Growth and shape transformations of giant phospholipid vesicles upon interaction with an aqueous oleic acid suspension

The interaction of two types of vesicle systems was investigated: micrometer-sized, giant unilamellar vesicles (GUVs) formed from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and submicrometer-sized, large unilamellar vesicles (LUVs) formed from oleic acid and oleate, both in a buffered aqueous solution (pH 8.8). Individual POPC GUVs were transferred with a micropipette into a suspension of oleic acid/oleate LUVs, and the shape changes of the GUVs were monitored using optical microscopy. The behavior of POPC GUVs upon transfer into a 0.8 mM suspension of oleic acid, in which oleic acid/oleate forms vesicular bilayer structures, was qualitatively different from the behavior upon transfer into a 0.3 mM suspension of oleic acid/oleate, in which oleic acid/oleate is predominantly present in the form of monomers and possibly non-vesicular aggregates. In both cases, changes in vesicle morphology were observed within tens of seconds after the transfer. After an initial increase of the vesicle cross-section, the vesicle started to evaginate, spawning dozens of satellite vesicles connected to the mother vesicle with narrow necks or tethers. In 60% of the cases of transfer into a 0.8 mM oleic acid suspension, the evagination process reversed and proceeded to the point where the membrane formed invaginations. In some of these cases, several consecutive transitions between invaginated and evaginated shapes were observed. In the remaining 40% of the cases of transfer into the 0.8 mM oleic acid suspension and in all cases of vesicle transfer into the 0.3 mM oleic acid suspension, no invaginations nor subsequent evaginations were observed. An interpretation of the observed vesicle shape transformation on the basis of the bilayer-couple model is proposed, which takes into account uptake of oleic acid/oleate molecules by the POPC vesicles, oleic acid flip-flop processes and transient pore formation.     

Lysozyme effect on oleic acid/oleate vesicles

We have analysed by means of turbidimetric, dynamic light scattering (DLS), and fluorimetric techniques the effect of lysozyme on negatively charged oleic acid/oleate vesicles. The addition of lysozyme brings about a decrease in optical density of the vesicle population, which finally results in a size distribution of oleate vesicles shifted toward smaller mean diameters. On the contrary, (a) when phosphatidylserine vesicles were used, lysozyme induces an increase of turbidity and a shift toward larger vesicle sizes; and (b) the addition of histone H1 or poly-L-lysine produces an aggregative behavior both in oleate and in phosphatidylserine vesicles. Experiments carried out with calcein-containing vesicles indicate that the observed changes in the lysozyme/oleate system occur with partial leakage of the vesicle content. All this is taken to suggest that the interaction between lysozyme and oleate vesicles is of quite specific nature, and certainly not just due to electrostatic interactions.     

A kinetic study of the growth of fatty acid vesicles

Membrane vesicles composed of fatty acids can be made to grow and divide under laboratory conditions, and thus provide a model system relevant to the emergence of cellular life. Fatty acid vesicles grow spontaneously when alkaline micelles are added to buffered vesicles. To investigate the mechanism of this process, we used stopped-flow kinetics to analyze the dilution of non-exchanging FRET probes incorporated into preformed vesicles during growth. Oleate vesicle growth occurs in two phases (fast and slow), indicating two pathways for the incorporation of fatty acid into preformed vesicles. We propose that the fast phase, which is stoichiometrically limited by the preformed vesicles, results from the formation of a "shell" of fatty acid around a vesicle, followed by rapid transfer of this fatty acid into the preformed vesicle. The slower phase may result from incorporation of fatty acid which had been trapped in an intermediate state. We provide independent evidence for the rapid transformation of micelles into an aggregated intermediate form after transfer from high to low pH. Our results show that the most efficient incorporation of added oleate into oleic acid/oleate vesicles occurs under conditions that avoid a large transient increase in the micelle/vesicle ratio.     

X-ray diffraction and spectroscopic studies of oleic acid-sodium oleate

The sodium oleate-oleic acid (1:1) complex (NaHOl(2)) is characterized using X-ray diffraction, FT-IR photoacoustic spectroscopy, FT-Raman spectroscopy, and DSC. The special arrangement of hydrogen-bonded pairs of carboxylic acid and carboxylate groups into unique "head-group" is supported by frequency shifts and partial or total disappearance of characteristic vibrations of carboxylic acid dimer and of carboxylate groups. The well-ordered state of hydrocarbon chains is demonstrated by the existence of sharp Raman bands in the C-C stretching region (1000-1150 cm-1) and other conformationally sensitive modes. The FT-Raman results suggest that the transition at about 32 degrees C involves the cooperative melting of methyl- and carboxyl/carboxylate-sided hydrocarbon chains. From the X-ray diffraction data it is clear that this transition is associated with the disintegration of the hydrogen-bonded carboxylate-carboxylic acid complex, followed by the separate formation of oleic acid and sodium oleate. The packing of hydrocarbon chain in the acid-soap complex is different from the parent oleic acid or sodium oleate. The hydrocarbon chains in the NaHOl(2) form more stable packing (O subcell) in comparison to that of oleic acid. A temperature composition phase diagram is presented.     

Solvent-free geranyl oleate production by enzymatic esterification

This study reports the maximization of geranyl oleate production by esterification of geraniol and oleic acid in a solvent-free system using a commercial lipase as catalyst. The operating conditions that maximized geranyl oleate production were determined to be 40 °C, geraniol to oleic acid molar ratio of 5:1, 150 rpm and 10 wt% of enzyme, with a resulting reaction conversion of about 93%. After determining the best reaction parameters, a kinetic study was performed and the results obtained in this step allow to conclude that an excess of alcohol (alcohol to acid molar ratio of 5:1), relatively low enzyme concentration (5 wt%) and temperature of 50 °C afforded nearly complete reaction conversion after 1 h of reaction. New experimental data on enzymatic esterification of geraniol and oleic acid for geranyl oleate production are reported in this work, showing a promising perspective of the technique to overcome the inconvenience of the chemical-catalyzed route.     

Synthesis of single- and double-13C-labeled cholesterol oleate

Cholesterol oleate with the 13C-label in oleic acid at the carbonyl and/or in the sterol ring at position 4 was synthesized by two methods: (1) cholesterol was condensed with oleic anhydride, prepared from [1-13C] oleic acid, in the presence of dimethylaminopyridine (DMAP) in anhydrous chloroform at room temperature for 4--5 h; (2) cholesterol or 13C-enriched cholesterol at position 4 were reacted with 90% [1-13C]-oleic acid in the presence of dicyclohexylcarbodiimide (DCC) and DMAP at room temperature in anhydrous chloroform for 1.25 h. The single-13C and double-13C-labeled cholesterol oleate were obtained in 90% yields after purification by silicic acid column chromatography. Their purity was assessed by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) and 13C-NMR spectroscopy. Tritium-labeled cholesterol oleate was also synthesized by method 1 using the fatty acid anhydride.     

Lipase-catalyzed reactions in vesicles as an approach to vesicle self-reproduction

Abstract

As a continuation of our endeavor to find conditions under which bounded aggregate structures are able to self-reproduce, we have investigated the reactivity of lipase, both in free solution and vesicle-entrapped, against mixed oleic acid/oleate/ethyl oleate vesicles. Three types of vesicles have been prepared and characterized: (A) oleic acid/oleate vesicles; (B) oleic acid/oleate/ethyl oleate vesicles; and (C) lipase containing oleic acid/oleate vesicles. Long time stability studies by quasi elastic light scattering show that whereas (B) and (C) vesicles remain stable with a diameter of 110-130 nm and monodisperse for over a period of one month, vesicles (A) separated from an initial single population of 105 nm diameter into two populations, having respectively 70 nm diameter (more than 95% of the particles) and 180-210 nm diameter (less than 5% of the total population). In the case of vesicles (C), it could be shown that the enzyme remains localized inside the vesicles and it does not protrude into the water bulk phase. The enzymatic hydrolysis of ethyl oleate (which is water-insoluble) incorporated in the B-vesicles was studied under two configurations: (I) by adding lipase externally to the B-vesicles; (II) by mixing vesicles (B) and vesicles (C). In both cases, the reaction progressed to 100% hydrolysis. In the first case, the reaction was attended by an increase of the number of vesicles, and since this hydrolysis reaction takes place within the boundary of the parent vesicles, the criteria of autopoietic self-reproduction of vesicles are satisfied. In the case (II) instead, no increase of the population number of particles could be detected. The possible reasons for this difference are discussed.     

Erratum to: Solvent-free geranyl oleate production by enzymatic esterification

This study reports the maximization of geranyl oleate production by esterification of geraniol and oleic acid in a solvent-free system using a commercial lipase as catalyst. The operating conditions that maximized geranyl oleate production were determined to be 40?°C, geraniol to oleic acid molar ratio of 5:1, 150?rpm and 10?wt% of enzyme, with a resulting reaction conversion of about 93%. After determining the best reaction parameters, a kinetic study was performed and the results obtained in this step allow to conclude that an excess of alcohol (alcohol to acid molar ratio of 5:1), relatively low enzyme concentration (5?wt%) and temperature of 50?°C afforded nearly complete reaction conversion after 1?h of reaction. New experimental data on enzymatic esterification of geraniol and oleic acid for geranyl oleate production are reported in this work, showing a promising perspective of the technique to overcome the inconvenience of the chemical-catalyzed route.     

Effect of sodium oleate on the hydrolysis of human plasma fibronectin by proteinases

Oleic acid binds in a saturable fashion to human plasma fibronectin (FN). Analysis of the binding indicated the presence of a high affinity binding site with nKa approximately equal to 10 uM-1. Furthermore, it was found that binding of sodium oleate to FN modulated its susceptibility to degradation by various proteinases. FN saturated with sodium oleate was hydrolysed at a higher rate by trypsin, cathepsin D, thermolysin and pancreatic elastase than native FN. In contrast, sodium oleate inhibits the activity of two human granulocyte proteinases, human leucocyte elastase (HLE) and cathepsin G on either FN or on their respective specific synthetic substrates (at concentrations ranging from 10(-6) mM to 10 mM). Cathepsin G inhibition was non-competitive and gave a Ki in the 10 uM range similar to the previously reported inhibitory constant of oleic acid toward HLE.     

Self-assembled vesicles of monocarboxylic acids and alcohols: conditions for stability and for the encapsulation of biopolymers

We tested the ability of saturated n-monocarboxylic acids ranging from eight to 12 carbons in length to self-assemble into vesicles, and determined the minimal concentrations and chain lengths necessary to form stable bilayer membranes. Under defined conditions of pH and concentrations exceeding 150 mM, an unbranched monocarboxylic acid as short as eight carbons in length (n-octanoic acid) assembled into vesicular structures. Nonanoic acid (85 mM) formed stable vesicles at pH 7.0, the pK of the acid in bilayers, and was chosen for further testing. At pH 6 and below, the vesicles were unstable and the acid was present as droplets. At pH ranges of 8 and above clear solutions of micelles formed. However, addition of small amounts of an alcohol (nonanol) markedly stabilized the bilayers, and vesicles were present at significantly lower concentrations (∼20 mM) at pH ranges up to 11. The formation of vesicles near the pK_a of the acids can be explained by the formation of stable RCOO⁻…HOOCR hydrogen bond networks in the presence of both ionized and neutral acid functions. Similarly, the effects of alcohols at high pH suggests the formation of stable RCOO⁻…HOR hydrogen bond networks when neutral RCOOH groups are absent. The vesicles provided a selective permeability barrier, as indicated by osmotic activity and ionic dye capture, and could encapsulate macromolecules such as DNA and a protein. When catalase was encapsulated in vesicles of decanoic acid and decanol, the enzyme was protected from degradation by protease, and could act as a catalyst for its substrate, hydrogen peroxide, which readily diffused across the membrane. We conclude that membranous vesicles produced by mixed short chain monocarboxylic acids and alcohols are useful models for testing the limits of stabilizing hydrophobic effects in membranes and for prebiotic membrane formation.     

Activation of protein kinase C by oleic acid. Determination and analysis of inhibition by detergent micelles and physiologic membranes: requirement for free oleate.

Sodium oleate is able to activate soluble protein kinase C (Murakami, K., Chan, S. Y., and Routtenberg, A. (1986) J. Biol. Chem. 261, 15424-15429) but is unable to activate membrane-bound enzyme (El Touny, S., Khan, W., and Hannun, Y. (1990) J. Biol. Chem. 265, 16437-16443). Because physiologic interactions of fatty acids with protein kinase C occur in the presence of membranes, the following studies were conducted to evaluate the effects of surfaces (detergent micelles or platelet membranes) on the activation of protein kinase C by oleate. At concentrations at or above the critical micellar concentration (CMC) of Triton X-100, oleate was present primarily in Triton X-100/oleate-mixed micelles, as determined by gel permeation chromatography and equilibrium dialysis binding studies. At concentrations slightly below the CMC for Triton X-100, the presence of oleate caused the formation of a limited number of mixed micelles. Studies of the dose-dependent activation of purified platelet protein kinase C by sodium oleate in the presence of different concentrations of Triton X-100 indicated that only unbound oleate was able to activate protein kinase C. Platelet protein kinase C was resolved into two major isoenzymes (types II (beta) and III (alpha)) which displayed nearly identical interaction with oleate. Activation of protein kinase C by oleate in a physiologic setting employing platelet substrates and endogenous platelet protein kinase C was investigated. Oleate potently activated protein kinase C in the cytosolic compartment. In platelet homogenates as well as in a reconstituted platelet cytosol and membrane system, the dose dependence of protein kinase C on oleate showed a significant shift to the right. Approximately 30% of oleate was associated with platelet cytosol and 70% was associated with platelet membranes. Partitioning of oleate into the two platelet compartments showed little change with pH, temperature, or duration of incubation. When corrected for free oleate concentration, activation of protein kinase C by oleate showed identical dose dependence in cytosol and homogenate. Arachidonate, a potential physiologic activator of protein kinase C, showed similar behavior as oleate although only 30% of arachidonate partitioned into platelet membranes with the majority of arachidonate (70%) remaining in the cytosolic fraction.(ABSTRACT TRUNCATED AT 400 WORDS)     

Production of 10-hydroxystearic acid from oleic acid by whole cells of recombinant Escherichia coli containing oleate hydratase from Stenotrophomonas maltophilia

A putative fatty acid hydratase from Stenotrophomonas maltophilia was cloned and expressed in Escherichia coli. The recombinant enzyme showed the highest hydration activity for oleic acid among the fatty acids tested, indicating that the enzyme is an oleate hydratase. The optimal conditions for the production of 10-hydroxystearic acid from oleic acid using whole cells of recombinant E. coli containing the oleate hydratase were pH 6.5, 35°C, 0.05% (w/v) Tween 40, 10 g l(-1) cells, and 50 g l(-1) oleic acid. Under these conditions, whole recombinant cells produced 49 g l(-1) 10-hydroxystearic acid for 4 h, with a conversion yield of 98% (w/w), a volumetric productivity of 12.3 g l(-1) h(-1), and a specific productivity of 1.23 g g-cells(-1) h(-1), which were 18%, 2.5-, and 2.5-fold higher than those of whole wild-type S. maltophilia cells, respectively. This is the first report of 10-hydroxystearic acid production using recombinant cells and the concentration and productivity are the highest reported thus far among cells.     

Production of a novel compound, 10,12-dihydroxystearic acid from ricinoleic acid by an oleate hydratase from Lysinibacillus fusiformis

A recombinant oleate hydratase from Lysinibacillus fusiformis converted ricinoleic acid to a product, whose chemical structure was identified as the novel compound 10,12-dihydroxystearic acid by gas chromatograph/mass spectrometry, Fourier transform infrared, and nuclear magnetic resonance analysis. The reaction conditions for the production of 10,12-dihydroxystearic acid were optimized as follows: pH?6.5, 30 °C, 15 g?l^?1 ricinoleic acid, 9 mg?ml^?1 of enzyme, and 4 % (v/v) methanol. Under the optimized conditions, the enzyme produced 13.5 g?l^?1 10,12-dihydroxystearic acid without detectable byproducts in 3 h, with a conversion of substrate to product of 90 % (w/w) and a productivity of 4.5 g?l^?1?h^?1. The emulsifying activity of 10,12-dihydroxystearic acid was higher than that of oleic acid, ricinoleic acid, stearic acid, and 10-hydroxystearic acid, indicating that 10,12-dihydroxystearic acid can be used as a biosurfactant.     

Enzymatic synthesis of cetyl oleate in a solvent-free medium using microwave irradiation and physicochemical evaluation

Abstract

A cosmetic ester, cetyl oleate was synthesized using microwave irradiated system. The esterification reaction was carried using Candida antarctica lipase B in a solvent-free media. The influence of various reaction parameters was studied, and the efficiency of Fermase CALB^TM10000 was compared with other enzymes. Equilibrium conversion of 97.5% was obtained within 20?min at 60?°C temperature, 1:2 oleic acid to cetyl alcohol molar ratio and 4% w/w dose of lipase. A comparative study showed that microwave irradiation is a much more efficient method than ultrasound irradiation and conventional heating. Fermase CALB^TM10000 was reusable over 6 enzymatic cycles as its stability improved under microwave system. Physicochemical parameters of cetyl oleate were tested in order to analyze its suitability for further cosmetic use.     

Ozonation of lysozyme in the presence of oleate in reverse micelles of sodium di-2-ethylhexylsulfosuccinate.

Ozone is shown to react with lysozyme in reverse micelles formed by 0.1 M sodium di-2-ethylhexylsulfosuccinate and 1.2-3 M water (pH 7.4) in isooctane solvent. The reaction of ozone is assessed by the oxidation of tryptophan residues in the protein to N-formylkynurenine. Cosolubilization of oleate in lysozyme-containing reverse micellar solutions at concentrations of 0.5-10 mM results in a progressive inhibition (19% to 82%) of the oxidation of tryptophan residues with a concentration for 50% inhibition around 2 mM. At this concentration of oleate, the magnitude of inhibition is independent of the micelle size and concentration, the overall interfacial area of reverse micelles, and the amount of ozone employed. These findings are discussed in terms of competitive reactions of ozone with unsaturated fatty acids and proteins in the lung lining fluid and in biological membranes.     

In vitro detachment of bacteria from ruminal digesta by buffered sodium oleate solutions

The effectiveness of a buffered sodium oleate solution was evaluated for detaching bacteria from ruminal digesta samples. A response surface derived from an octagonal design was used to determine the pH and concentration combination for maximum detachment of total and cellulolytic bacteria. The total number of bacteria detached increased up to 81% over control with treatment of a pH 8.8 and 1.5% sodium oleate solution. The recovery of cellulolytic bacteria was decreased to 35% of control with treatment of a pH 9.0 and 0.1% sodium oleate solution. Attempts to improve the recovery of viable bacteria exposed to sodium oleate solutions were unsuccessful. This response surface design identified an optimal pH and concentration that were consistent with existing information regarding detachment of total bacteria, and suggested that sodium oleate, at the concentrations tested, was toxic to the cellulolytic population of the rumen.     

Fatty acid alcohol ester-synthesizing activity of lipoprotein lipase

The fatty acid alcohol ester-synthesizing activity of lipoprotein lipase (LPL) was characterized using bovine milk LPL. Synthesizing activities were determined in an aqueous medium using oleic acid or trioleylglycerol as the acyl donor and equimolar amounts of long-chain alcohols as the acyl acceptor. When oleic acid and hexadecanol emulsified with gum arabic were incubated with LPL, palmityl oleate was synthesized, in a time- and dose-dependent manner. Apo-very low density lipoprotein (apoVLDL) stimulated LPL-catalyzed palmityl oleate synthesis. The apparent equilibrium ratio of fatty acid alcohol ester/oleic acid was estimated using a high concentration of LPL and a long (20 h) incubation period. The equilibrium ratio was affected by the incubation pH and the alcohol chain length. When the incubation pH was below pH 7.0 and long chain fatty acyl alcohols were used as substrates, the fatty acid alcohol ester/free fatty acid equilibrium ratio favored ester formation, with an apparent equilibrium ratio of fatty acid alcohol ester/fatty acid of about 0.9/0.1. The equilibrium ratio decreased sharply at alkaline pH (above pH 8.0). The ratio also decreased when fatty alcohols with acyl chains shorter than dodecanol were used. When a trioleoylglycerol/fatty acyl alcohol emulsion was incubated with LPL, fatty acid alcohol esters were synthesized in a dose- and time-dependent fashion. Fatty acid alcohol esters were easily synthesized from trioleoylglycerol when fatty alcohols with acyl chains longer than dodecanol were used, but synthesis was decreased with fatty alcohols with acyl chain lengths shorter than decanol, and little synthesizing activity was detected with shorter-chain fatty alcohols such as butanol or ethanol.     

Relation between Ca2+ uptake and fluidity of brush-border membranes isolated from rabbit small intestine and incubated with fatty acids and methyl oleate

The rate of incorporation of oleic acid into isolated brush-border membranes was found to be considerably faster than methyl oleate incorporation under similar experimental conditions. The effects of fatty acids and methyl oleate incorporation on Ca2+ uptake and fluidity were monitored. Whereas treatment with 0.01-0.05 mM oleic acid corresponding to incorporations smaller than 90 nmol/mg protein enhanced Ca2+ transport, exposures to higher concentrations of this fatty acid corresponding to incorporations larger than 150 nmol/mg protein, decreased uptake of this cation. On the other hand, treatment with 0.01-0.2 mM methyl oleate corresponding to incorporations of up to 220 nmol/mg protein had only a stimulatory effect on the Ca2+ uptake. Oleic acid, linoleic acid and methyl oleate decreased the fluorescence anisotropy of membranes labelled with diphenylhexatriene in a dose-dependent manner. In contrast, palmitic acid had little or no effect on the diphenylhexatriene-reportable order of the membrane within the range of concentrations used. Monitored as a function of temperature, the anisotropy values showed a gradual melting for both the control and lipid-treated membranes. The results support the concept that saturated and cis-unsaturated fatty acids dissolve in different lipid domains and this in itself appears to be an important factor defining whether the biological function of the membrane is affected by the uptake. Incorporation of cis-unsaturated fatty acids in domains harboring the Ca2+ uptake process increases Ca2+ uptake in concert with increased diphenylhexatriene-monitored fluidity. However, when concentrations of such fatty acids in these domains become sufficiently great, the presence of a largely increased number of free carboxyl groups at the membrane surface causes inhibition of Ca2+ uptake.