High phosphatidylcholine weights compared to lysophosphatidylcholine weights, in micellar intestinal contents, in the rat (author's transl)

Casein hydrolysat, lactose and lipids (100 mg of fatty acids) were introduced in the stomach of rats by a gastric tube: either pure tri-oleoylglycerol, or phospholipids, or phosphatidylcholines, or the mixture 9/1 to fatty acid weight of tri-oleoylglycerol-phospholipids or phosphatidylcholines. The rats were killed 2 h later. The intraluminal intestinal lipids of the oil and micellar phases were separated after microfiltration (Millipore filters) in preference to the filtration by gel chromatography on polyacrylamide agarose, as an hydrolysis of intraluminal phospholipid occurred after the column elution. 1. After a quantitative recovery of the intestinal lipids (no separation of the oil and micellar phases), a strong hydrolysis of the tri-oleoyglycerol was observed; in opposition, large amounts of intact phospholipids appeared. 2. After isolation of the micellar phases, no triglycerides were recovered, but fatty acids and partial glycerides from the hydrolysed tri-oleoylglycerol and dietary phosphatidylcholines and small quantities of lyso-phosphatidylcholines (hydrolysed forms) were present. 3. After ingestion of the tri-oleoylglycerol as lipid dietary source, the intestinal micellar phases contained endogenous phosphatidylcholines and a few amounts of lysophosphatidylcholines, which had mainly bile origin, since the fatty acid composition of these micellar phosphatidylcholines approached the bile phosphatidylcholine fatty acid composition. The micellar lysophosphatidylcholine masses represented one-fourth of the micellar phosphatidylcholine masses. 4. In these experiments the phosphatidylcholine lysophosphatidylcholine ratio was always high: this means that small quantities of exogenous and endogenous lysophosphatidylcholines appeared in the micellar phases.     

Short-chain phospholipids as detergents

The physico-chemical properties of short-chain phosphatidylcholine are reviewed to the extent that its biological activity as a mild detergent can be rationalized. Long-chain diacylphosphatidylcholines are typical membrane phospholipids that form preferentially smectic lamellar phases (bilayers) when dispersed in water. In contrast, the preferred phase of the short-chain analogues dispersed in excess water is the micellar phase. The preferred conformation and the dynamics of short-chain phosphatidylcholines in the monomeric and micellar state present in H(2)O are discussed. The motionally averaged conformation of short-chain phosphatidylcholines is then compared to the single-crystal structures of membrane lipids. The main conclusion emerging is that in terms of preferred conformation and motional averaging short-chain phosphatidylcholines closely resemble their long-chain analogues. The dispersing power of short-chain phospholipids is emphasized in the second part of the review. Evidence is presented to show that this class of compounds is superior to most other detergents used in the solubilization of membrane proteins and the reconstitution of the solubilized proteins to artificial membrane systems (proteoliposomes). The prominent feature of the solubilization/reconstitution of integral membrane proteins by short-chain PC is the retention of the native protein structure and hence the protein function. Due to their special detergent-like properties, short-chain PC lend themselves very well not only to membrane solubilization but also to the purification of integral membrane proteins. The retention of the native protein structure in the solubilized state, i.e. in mixed micelles consisting of the integral membrane protein, intrinsic membrane lipids and short-chain PC, is rationalized. It is hypothesized that short-chain PC interacts primarily with the lipid bilayer of a membrane and very little if at all with the membrane proteins. In this way, the membrane protein remains associated with its preferred intrinsic membrane lipids and retains its native structure and its function.     

Micellar enzymology

Experimental approaches to modelling the enzymatic function of biological membranes are discussed. Emphasis is given to pseudohomogeneous systems such as proteolipid complexes and enzymes in organic solvents; the latter are solubilized with phospholipids or synthetic surfactants. Methods for producing and studying such micellar systems are considered. The key research problems of micellar enzymology are formulated and its relation to enzyme membranology is discussed. Finally, the new potentialities are noted of applied enzymology (biotechnology) offered by application of a colloidal solution of water in organic solvents as a microheterogeneous medium for enzymatic reactions.     

Micellar enzymology: potentialities in fundamental and applied areas

Micellar enzymology, a new trend in molecular biology, studies catalysis by enzymes entrapped in hydrated reversed micelles of surfactants (phospholipids, detergents) in organic solvents. In this review, the key research problems of micellar enzymology are formulated and examples of biocatalysis in microheterogeneous media are discussed. In particular, new applications are presented of micellar enzymology in fine organic syntheses, in clinical and chemical analyses (bioluminescence and enzyme immunoassays), in bioconversion of energy and mass, in therapy (engineering of new drugs capable of targeted penetration into cells), as well as in biotechnology (processes using nanogranulated or nanocapsulated enzymes).     

The second E.C. Slater lecture. Micellar enzymology: its relation to membranology

Micellar enzymology, a new trend in molecular biology, studies catalysis by enzymes entrapped in hydrated reversed micelles composed of surfactants (phospholipids, detergents) in organic solvents. The key research problems of micellar enzymology and its relation to enzyme membranology are discussed.     

Improvement in extraction and catalytic activity of Mucor javanicus lipase by modification of AOT reverse micelle

Reverse micelles are formed in apolar solvents by spontaneous aggregation of surfactants. Surfactant sodium bis (2-ethylhexyl) sulfosuccinate (AOT) is most often used for the reverse micellar extraction of enzymes. However, the inactivation of enzyme due to strong interaction with AOT molecules is a severe problem. To overcome this problem, the AOT/water/isooctane reverse micellar system was modified by adding short chain polyethylene glycol 400 (PEG 400). The modified AOT reverse micellar system was used to extract Mucor javanicus lipase from the aqueous phase to the reverse micellar phase. The extraction efficiency (E) increased with the increase in PEG 400 addition and the maximum E in PEG 400 modified system was twofold higher than that in the PEG 400-free system. Upon addition of PEG 400, the water activity (a(w)) of aqueous phase decreased, whereas a(w) of reverse micellar phase increased. The circular dichroism spectroscopy analysis revealed that PEG 400 changes the secondary and tertiary structure of lipase. The maximum specific activity of lipase extracted in PEG 400-modified reverse micellar system was threefold higher than that in the PEG-free system.     

Purification and concentration of alkaline phosphatase by selective permeabilization of Escherichia coli using reverse micellar solutions

Recovery of alkaline phosphatase (AP) from the periplasm of Escherichia coli using reverse micellar solutions (RMSs) of sodium dioctyl sulfosuccinate (AOT) in aliphatic hydrocarbons has been attempted. A variety of surface-active agents, solvents, and reverse micellar conditions were screened, and an excellent recovery of the enzyme in a concentrated form, with a high purification factor, was obtained in a single-step process. The permeabilization process strongly depended on the water content of the RMS as well as on the amount of water coating the microbial cell surface. The product was almost free from nucleic acids. In addition, because of the low affinity of AOT and the organic solvent for the aqueous phase, contamination by the permeabilizing agents would also be negligible.     

Kinetics of <Emphasis Type="Italic">p</Emphasis>-nitrophenyl Acetate Hydrolysis Catalyzed by <Emphasis Type="Italic">Mucor javanicus</Emphasis> Lipase in AOT Reverse Micellar Solutions Formulated in Different Organic Solvents

The rate of hydrolysis of p-nitrophenyl acetate (PNPA) catalyzed by Mucor javanicus lipase has been measured in AOT reverse micellar solutions formulated in aliphatic hydrocarbons, aromatic hydrocarbons and a chlorinated compound. The study has been performed at a single value of W = ([water]/[AOT]) = 6.0. Fluorescence decay measurements of intrinsic enzyme fluorescence, mainly due to tryptophan residues, in the different reverse micellar systems were also carried out, in an attempt to obtain some insight on the effect of the organic solvent on the protein conformation. Differences observed in the kinetics of the fluorescence decays of tryptophan residues of the lipase incorporated to the micelles with the different external organic solvents were also found in the catalytic behaviour of the enzyme. In particular, it is observed that the contribution of the long lived component of the fluorescence decay is considerably higher (ca. 40%) in aliphatic than in aromatic solvents (ca. 15%), indicating significant differences in the protein conformation. This effect of the organic solvent on the protein conformation is also observed in the enzymatic activity, which is higher in the aromatic than in the aliphatic solvents.     

Cholera toxin modifies diverse GTP-modulated regulatory proteins

Using thin layer chromatography (TLC) and various colorimetric procedures, the exometabolite of $\text{Leishmania}\text{donovani}$ was shown to be a novel glycopeptidophosphosphingolipid. In aqueous medium the exometabolite aggregated to form micellar structures of high molecular weight. Purity of the various preparations and the novel nature of the micellar structures was demonstrated by TLC. These micelles are unique because they do not break up upon solvation in organic solvents. This indicates that once the supramolecular structure is established, its integrity is maintained by forces other than the apolar ones involved in its formation.     

Polarographic measurement of oxygen uptake using lipoxygenase in reverse micelles

Lipoxygenase-catalyzed linoleic acid peroxidation was chosen as a model system to study the applicability of oxygraphy to monitor the oxygen uptake in organic solvents containing reverse micelles. Care was taken to control the oxygen back transfer from the atmosphere to the sample micellar solution, resulting in a significant improvement of electrode response. Under these conditions, lipoxygenase activity was linear up to 100 mug of enzyme. Given the quality of the calibration curve and the good correlation between lipoxygenase and ascorbate oxidase, the described technique is proposed as an alternative method for determining lipoxygenase activity in reverse micelles. The reliability of this technique was confirmed by the good agreement between polarography and classic spectrophotometry in kinetic studies. Preliminary experiments carried out on soybean cells solubilized in a Tween 85-isopropylpalmitate system demonstrated that a light-dependent oxygen uptake can be measured. The authors propose that the Clark-type electrode be employed to study both the activity of oxidasic enzymes in reverse micelles and cell viability and physiology in organic solvents.     

Liquid-liquid extraction of alpha-lactalbumin using reverse micellar organic solvent

The liquid-liquid extraction of alpha-lactalbumin based on reverse micellar organic solvents was investigated. Forward extraction of the protein in the reverse micellar organic phase from aqueous feed solutions was strongly dependent on the initial pH of the feed solution and the complete forward extraction of 0.03 mM alpha-lactalbumin was successfully achieved at pH 6.0. The forward extraction percentage steeply decreased with increasing KCl concentration, while in the NaCl system the forward extraction was independent on the salt concentration below 1 M. From the circular dichroic measurement, higher order structure of the recovered alpha-lactalbumin through the extraction process was well preserved.     

The role of phospholipid acyl chains in the activation of mitochondrial ATPase complex.

1. The role of length and unsaturation of phospholipid acyl chains in the activation of ATPase complex was studied with synthetic phosphatidylcholines and a phospholipid-dependent preparation obtained after cholate-extraction of submitochondrial particles (Kagawa, Y. and Racker, E. (1966) J. Biol. Chem. 241, 2467--2474). 2. Micelle-forming, short-chain phosphatidylcholines produced activation only at critical micellar concentration. The reactivated complex was cold-stable but the oligomycin sensitivity was low. 3. Bilayer-forming saturated phosphatidylcholines produced activation which was maximal at 9 carbon atoms in each chain but decreased sharply as the chain-length was increased and essentially disappeared at 14 carbon atoms. By contrast the oligomycin-sensitivity increased with the increase in chain length. 4. Activation of ATPase complex reappeared when bilayers were formed with long-chain unsaturated phosphatidylcholines. The activity was oligomycin sensitive. Significant inhibition of activity was observed also after incorporation of cholesterol into the bilayers. 5. By contrast the activation induced by negatively charged liposomes of diacylphosphatidylglycerol was independent on acyl-chain composition and occurred at very low amounts of phospholipid. 6. The discontinuity in the Arrhenius plot of activity of the ATPase complex reactivated with saturated phospholipids was found at temperatures close to the gel-to-liquid crystalline transition of the lipid showing that the activity of ATPase complex was sensitive to the physical state of membrane phospholipids. 7. It is concluded that (a) reactivation of ATPase complex by isoelectric phospholipids is an interfacial activation, the minimum requirement for the lipid effect being micelle formation. (b) In order to gain the properties of the native complex a stable lamellar phase is needed. Both activity and oligomycin sensitivity are regulated by the chain length and degree of unsaturation of phospholipid acyl chains.     

Dynamics of Phospholipid Content in the Vacuolar Membrane of Red Beet Taproots Exposed to Abiotic Stress

The composition of vacuolar membrane phospholipids in the taproot of red beet (Beta vulgaris L.), cv. Modana, was determined at normal conditions and under different types of stress (hypo- and hyperosmotic and oxidative stress). The experiments have shown that, among vacuolar membrane phospholipids in red beet taproot, phosphatidylcholines and phosphatidylethanolamines dominated and accounted for 70% of total phospholipids. It is interesting that the content of phosphatidic acid was high (20% of total phospholipids of the vacuolar membrane). Stress effects brought about changes in the composition of membrane phospholipids, which may be an element of phenotypic adaptation. Under hypoosmotic stress, reliable changes in the content of phosphatidic acid were observed, hyperosmotic stress was associated with changes in the level of phosphatidylcholines and phosphatidylinositols, and oxidative stress was notable for changes in the content of phosphatidylethanolamines and phosphatidylserines. The most significant changes were observed in the classes of phospholipids that may be involved in structural modification of membranes associated with transformation of their bilayer lamellar structure into hexagonal. These phospholipids comprise phosphatidic acid, phosphatidylcholines, and phosphatidylethanolamines. Revealed changes in the content of these phospholipids may alter the ratio between lamellar bilayer and nonbilayer hexagonal lipid structures in the vacuolar membrane and act as an important adaptation mechanism ensuring protection against stress.     

Stability of a Pseudomonas Sp. Lipase:Comparison Between Solubilized Enzyme in Reverse Micelles and Suspended Lipase in Dry Solvents

The stability of a relatively hydrophobic lipase from Pseudomonas sp., solubilized in reverse micellar media or suspended in dry solvents, was studied and compared. Factors such as the enzyme-solvent interaction, enzyme environment, hydration degree of the system, interphase quality, droplet size, and water activity were studied. A mixed micellar system which stabilized the lipase is reported. In the case of simple AOT micelles, lipase destabilization with respect to water in small droplet sizes and stabilization in the biggest micelles was observed. These effects resulted from lipase penetration into the interphase of the smaller nanodroplets, and the restriction of its conformational mobility in the region of structured water of the largest micelles, respectively. Mixed micelles increased lipase stability, which was mainly related to increased droplet size. Modification with polyethylene glycol decreased lipase stability in reverse micelles, due to the greater interaction with the micellar interphase. The preparation of nanodroplets, in which native and modified lipases were 5.4 and 9.4 times, respectively, more stable than in water, is reported. In contrast to the micellar media, low water contents (low A_w values) stabilized the solid lipase suspended in organic solvent systems. Under the hydration conditions studied here, lipase stability increased when more polar solvents were used. Two alternatives were necessary to obtain similar stabilities in n-heptane as compared with polar solvents: reduction of the water content or use of a low aquaphilic support.     

Storage of Pollen Grains in Organic Solvents: Effect of Organic Solvents on Leaching of Phospholipids and its Relationship to Pollen Viability

In vitro germinability and membrane integrity (as revealed bythe fluorochromatic reaction (FCR) test) of pollen grains ofCrotalaria retusa L. stored in various organic solvents forsix months at –20±2 °C were studied and correlatedwith leaching of lipids, phospholipids, sugars and free aminoacids from pollen grains into organic solvents during storage.Pollen grains stored in organic solvents with low dielectricconstants (a measure of their non-polar nature), such as hexane,cyclohexane and diethyl ether, showed high scores for germinationand FCR and very little leaching of phospholipids, sugars andamino acids. Pollen grains stored in solvents with high dielectricconstants (a measure of their polar nature) such as isopropanoland methanol did not show germination or positive FCR scores,but showed extensive leaching of phospholipids, sugars and freeamino acids. The viability of pollen grains stored in organicsolvents seems to be determined largely by the effect of theorganic solvents on pollen phospholipid composition, which inturn affects membrane integrity and consequently pollen viability. Crotalaria retusa, organic solvents, pollen storage, viability, phospholipids     

A comparison of lipase-catalyzed ester hydrolysis in reverse micelles, organic solvents, and biphasic systems

The performance of lipases from Candida rugosa and wheat germ have been investigated in three reaction media using three acetate hydrolyses as model reactions (ethyl acetate, allyl acetate, and prenyl acetate). The effect of substrate properties and water content were studied for each system (organic solvent, biphasic system, and reverse micelles). Not unexpectedly, the effect of water content is distinct for each system, and the optimal water content for enzyme activity is not always the same as that for productivity. A theoretical model has been used to simulate and predict enzyme performance in reverse micelles, and a proposed partitioning model for biphasic systems agrees well with experimental results. While the highest activities observed were in the micellar system, productivity in microemulsions is limited by low enzyme concentrations. Biphasic systems, however, support relatively good activity and productivity. The addition of water to dry organic solvents, combined with the dispersion of lyophilized enzyme powders in the solvent, resulted in significant enzyme aggregation, which not surprisingly limits the applicability of the "anhydrous" enzyme suspension approach. (c) 1995 John Wiley & Sons, Inc.     

Preferable stimulation of PON1 arylesterase activity by phosphatidylcholines with unsaturated acyl chains or oxidized acyl chains at sn-2 position

To examine the effect of phospholipids on PON1 activities, purified PON1 was exposed to phospholipids prior to the determination of arylesterase and paraoxonase activities. Phosphatidylcholines with saturated acyl chains (C10-C16) showed a stimulation of both activities, chain length-dependent, with a greater stimulation of arylesterase activity, suggesting the implication of lipid bilayer in the stimulatory action. Such a preferable stimulation of arylesterase activity was more remarkable with phosphatidylcholines with polyunsaturated acyl chains or oxidized chains at sn-2 position, implying that the packing degree of acyl chain may be also important for the preferable stimulation of arylesterase activity. Separately, 1-palmitoyl-lysoPC also stimulated arylesterase activity preferably, indicating that the micellar formation of lipids around PON1 also contributes to the stimulatory action. Additionally, phosphatidylglycerols slightly enhanced arylesterase activity, but not paraoxonase activity. In contrast, phosphatidylserine and phosphatidic acid (≥0.1 mM) inhibited both activities Further, such a preferable stimulation of arylesterase activity by phosphatidylcholines was also reproduced with VLDL-bound PON1, although to a less extent. These data indicate that phosphatidylcholines with polyunsaturated acyl chains or oxidized chain, or lysophosphatidylcholine cause a preferable stimulation of arylesterase activity, thereby contributing to the decrease in the ratio of paraoxonase activity to arylesterase activity.     

Preferable stimulation of PON1 arylesterase activity by phosphatidylcholines with unsaturated acyl chains or oxidized acyl chains at sn-2 position

To examine the effect of phospholipids on PON1 activities, purified PON1 was exposed to phospholipids prior to the determination of arylesterase and paraoxonase activities. Phosphatidylcholines with saturated acyl chains (C10-C16) showed a stimulation of both activities, chain length-dependent, with a greater stimulation of arylesterase activity, suggesting the implication of lipid bilayer in the stimulatory action. Such a preferable stimulation of arylesterase activity was more remarkable with phosphatidylcholines with polyunsaturated acyl chains or oxidized chains at sn-2 position, implying that the packing degree of acyl chain may be also important for the preferable stimulation of arylesterase activity. Separately, 1-palmitoyl-lysoPC also stimulated arylesterase activity preferably, indicating that the micellar formation of lipids around PON1 also contributes to the stimulatory action. Additionally, phosphatidylglycerols slightly enhanced arylesterase activity, but not paraoxonase activity. In contrast, phosphatidylserine and phosphatidic acid (> or =0.1 mM) inhibited both activities Further, such a preferable stimulation of arylesterase activity by phosphatidylcholines was also reproduced with VLDL-bound PON1, although to a less extent. These data indicate that phosphatidylcholines with polyunsaturated acyl chains or oxidized chain, or lysophosphatidylcholine cause a preferable stimulation of arylesterase activity, thereby contributing to the decrease in the ratio of paraoxonase activity to arylesterase activity.     

A new way in homogeneous immunoassay: reversed micellar systems as a medium for analysis

Possibilities of a new principle for the homogeneous enzyme immunoassay utilizing the systems of surfactant reversed micelles in organic solvents have been demonstrated taking thyroxine determination as an example. The catalytic activity of an enzyme, solubilized in such systems, is determined by the ratio of geometric dimensions of the micellar matrice and the enzyme molecule. The addition of antibodies against thyroxine to the peroxidase-thyroxine conjugate, solubilized in the system of reversed micelles of aerosol OT in octane, leads to the formation of the immune complex whose size differs substantially from that of the initial enzyme-antigen conjugate. This induces changes in the peroxidase catalytic activity. The addition of free thyroxine to the system stimulates the conjugate release from the immune complex and, consequently, the reduction of the peroxidase activity to the initial level. Sensitivity of the analysis in reversed micellar systems can be regulated by changing the surfactant hydration degree. Substances of different nature (both hydrophobic and hydrophylic) can be solubilized in reverse micellar systems under standard conditions, which allows determination of water insoluble antigens.     

Morphology of self‐assembled structures formed by short peptides from the amyloidogenic protein tau depends on the solvent in which the peptides are dissolved

The aggregation behavior of peptides Ac‐VQIVYK‐amide (AcPHF6) and Ac‐QIVYK‐amide (AcPHF5) from the amyloidogenic protein tau was examined by atomic force microscopy (AFM) and fluorescence microscopy. Although AcPHF5 did not show enhancement of thioflavin T (ThT) fluorescence in aqueous buffer, distinct aggregates were discernible when peptide was dissolved in organic solvents such as methanol (MeOH), trifluoroethanol (TFE), and hexafluoroisopropanol (HFIP) dried on mica and examined by AFM. Self‐association was evident even though the peptide did not have the propensity to form secondary structures in the organic solvents. In dried films, the peptide adopts predominantly β‐conformation which results in the formation of distinct aggregates. ThT fluorescence spectra and fluorescence images indicate the formation of fibrils when AcPHF6 solutions in organic solvents were diluted into buffer. AcPHF6 had the ability to organize into fibrillar structures when AFM samples were prepared from peptide dissolved in MeOH, TFE, HFIP, and also when diluted into buffer. AcPHF6 showed propensity for β‐structure in aqueous buffer. In MeOH and TFE, AcPHF6 showed helical and β‐structure. Morphology of the fibrils was dependent on peptide conformation in the organic solvents. The structures observed for AcPHF6 are formed rapidly and long incubation periods in the solvents are not necessary. The structures with varying morphologies observed for AcPHF5 and AcPHF6 appear to be mediated by surfaces such as mica and the organic solvents used for dissolution of the peptides. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.