Amyloglucosidase enzymatic reactivity inside lipid vesicles

Efficient functioning of enzymes inside liposomes would open new avenues for applications in biocatalysis and bioanalytical tools. In this study, the entrapment of amyloglucosidase (AMG) (EC 3.2.1.3) from Aspergillus niger into dipalmitoylphosphatidylcholine (DPPC) multilamellar vesicles (MLVs) and large unilamellar vesicles (LUVs) was investigated. Negative-stain, freeze-fracture, and cryo-transmission electron microscopy images verified vesicle formation in the presence of AMG. Vesicles with entrapped AMG were isolated from the solution by centrifugation, and vesicle lamellarity was identified using fluorescence laser confocal microscopy. The kinetics of starch hydrolysis by AMG was modeled for two different systems, free enzyme in aqueous solution and entrapped enzyme within vesicles in aqueous suspension. For the free enzyme system, intrinsic kinetics were described by a Michaelis-Menten kinetic model with product inhibition. The kinetic constants, V _max and K _m , were determined by initial velocity measurements, and K _i was obtained by fitting the model to experimental data of glucose concentration-time curves. Predicted concentration-time curves using these kinetic constants were in good agreement with experimental measurements. In the case of the vesicles, the time-dependence of product (glucose) formation was experimentally determined and simulated by considering the kinetic behavior of the enzyme and the permeation of substrate into the vesicle. Experimental results demonstrated that entrapped enzymes were much more stable than free enyzme. The entrapped enzyme could be recycled with retention of 60% activity after 3 cycles. These methodologies can be useful in evaluating other liposomal catalysis operations.     

Preparation and applications of biofunctionalized,supported lipid bilayers and vesicles

Biofunctional surfaces require advanced design and preparation to match the (bio)recognition ability of biological systems [1]. This requires combined topographic, chemical and visco-elastic surface patterns to match proteins at the nm scale and cells at the micrometer scale. One example of biochemical functionalization, presented here, and which is of both fundamental and application interest, is supported biomimectic (cell)membranes. Specifically we describe preparation and applications of supported phospholipid membranes, which can be made on certain surfaces from unilamellar, 25–200 nm vesicles. On SiO2 at normal pH and with neutral lipids, the vesicles first adsorb intact, and then undergo a phase transformation to a supported bilayer. We have studied the coverage-, vesicle size-, and T-dependence of this process [2], using QCM-D [3], AFM, and SPR. When SiO2 is replaced by TiO2, vesicles adsorb intact. A surface pre-covered with intact vesicles, can be AFM patterned into areas with bilayer, vesicles, and empty surface patches [4]. The results depend critically on AFM tip interaction with vesicle and bilayer, which has been modeled by Monte Carlo simulations [5]. These biomembranes are inert towards protein adsorption [6] and cell attachement [7], which opens up for various applications. Addition of functional molecules, allows sensor functions [8]. Another application is functionalized membranes for surface-specific (stem) cell interactions [9].     

Large-scale preparation of asymmetrically labeled fluorescent lipid vesicles

A method for producing lipid vesicles containing fluorescent phospholipid analogues localized to the inner leaflet of their membrane was developed. Incubation of a 450-fold molar excess of serum albumin with lipid vesicles symmetrically labeled with 1 mol % 1-palmitoyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazolyl)amino-caproyl phosphatidylcholine resulted in the removal of 99% of the fluorescent lipid from the outer leaflet. Asymmetrically labeled vesicles were separated from albumin/lipid complexes by gel filtration chromatography. Vesicles prepared in this manner were unable to transfer fluorescent lipid to cells during liposome-cell incubations. Liposomes asymmetrically labeled with other 4-nitrobenzo-2-oxa-1,3-diazole (NBD)-phospholipid analogues were also prepared. Removal of amino-dodecanoyl-NBD-labeled lipids from the outer leaflet of liposomes required three times more bovine serum albumin, and 48 h of incubation. This method can be used to produce large amounts of asymmetrically labeled liposomes suitable for use in investigating a variety of membrane phenomena.     

Polymerized lipid vesicles as colorimetric biosensors for biotechnological applications

Supramolecular chemical assemblies composed of polydiacetylene (PDA) exhibit rapid colorimetric transitions upon specific interactions with a variety of biological analytes in aqueous solutions. Among the analytes that give rise to the unique blue-red color changes are lipophilic enzymes, antibacterial peptides, ions, antibodies, and membrane penetration enhancers. The chemical assemblies include conjugated PDA, responsible for the chromatic transitions, and the molecular recognition elements, which are either chemically or physically associated with the PDA. Thus, by incorporation of specific recognition elements, the system can be designed in ways allowing for highly selective identification of analytes. In particular, receptors and epitopes can be incorporated within the sensor assembly, which then determine the specificity of the colorimetric transitions. The PDA-based molecular assemblies are robust and can be readily applied to diagnosis of physiological molecules and for rapid screening of chemical and biological libraries, for example, in 96 well-plate platforms.     

Specific reactivity of lipid vesicles conjugated with oriented anti-lactose antibody fragments

The method previously described (Sinha, D. and Karush, F. (1979) Biochem. Biophys. Res. Commun. 90, 554--560) for the oriented attachment of immunoglobulins to lipid vesicles has been used to confer specific reactivity on liposomes by their conjugation with anti-lactose Fab' fragments derived from rabbit IgG antibody. It is estimated that one-third of the Fab' fragments was irreversibly attached to liposomal membrane, resulting in a membrane concentration of 2 mmol of Fab' per mol of total lipid. The specific reactivity of the modified liposomes was demonstrated by agglutination with a multivalent, lactose-containing diheteroglycan. The availability of virtually all of the binding sites of the attached antibody for reaction with ligand was established by a fluorescence quenching titration with N-(N epsilon-Dnp-L-lysyl)-p-aminophenyl-beta-lactoside. An intrinsic association constant of 8.9 x 10(6) M-1 was found for the attached Fab' compared to a value of 2.8 x 10(6) M-1 for free anti-lactose Fab'. In addition the maximum values for the quenching by bound ligand of the fluorescence of free and attached antibody were the same. It can be concluded that the chemical procedures used to effect attachment of the antibody to the lipid vesicles allow retention of the original structure of the antibody site and its accessibility to external components.     

The surfactant lipid transporter ABCA3 is N-terminally cleaved inside LAMP3-positive vesicles

ABCA3 mutations cause fatal surfactant deficiency and interstitial lung disease. ABCA3 protein is a lipid transporter indispensible for surfactant biogenesis and storage in lamellar bodies (LB). The protein folds in endoplasmic reticulum and is glycosylated in Golgi en route to the membrane of mature LB and their precursor multivesicular bodies (MVB). In immunoblots, C-terminally labeled ABCA3 appears as two protein bands of 150 and 190 kDa. Using N- and C-terminal protein tags and hindering ABCA3 processing we show that the 150 kDa protein represents the mature ABCA3 whose N-terminus is cleaved by a cysteine protease inside MVB/LB.

Structured summary

MINT-7996633: Calnexin (uniprotkb:P27824) and ABCA3 (uniprotkb:Q99758) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7996380, MINT-7996593, MINT-7996607: LAMP3 (uniprotkb:Q9UQV4) and ABCA3 (uniprotkb:Q99758) colocalize (MI:0403) by fluorescence microscopy (MI:0416)     

Membrane-active peptides: Binding,translocation, and flux in lipid vesicles

Recently, new and improved methods have been developed to measure translocation of membrane-active peptides (antimicrobial, cytolytic, and amphipathic cell-penetrating peptides) across lipid bilayer membranes. The hypothesis that translocation of membrane-active peptides across a lipid bilayer is determined by the Gibbs energy of insertion of the peptide into the bilayer is re-examined in the light of new experimental tests. The original hypothesis and its motivation are first revisited, examining some of the specific predictions that it generated, followed by the results of the initial tests. Translocation is understood as requiring two previous steps: binding and insertion in the membrane. The problem of peptide binding to membranes, its prediction, measurement, and calculation are addressed. Particular attention is given to understanding the reason for the need for amphipathic structures in the function of membrane-active peptides. Insertion into the membrane is then examined. Hydrophobicity scales are compared, and their influence on calculations is discussed. The relation between translocation and graded or all-or-none peptide-induced flux from or into lipid vesicles is also considered. Finally, the most recent work on translocation is examined, both experimental and from molecular dynamics simulations. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.     

Peering inside lipid rafts and caveolae

Clustering of proteins into membrane microdomains, such as lipid rafts and caveolae, could act as a mechanism for regulating cell signaling and other cellular functions. Certain lipid modifications are hypothesized to target proteins to these domains on the cytoplasmic leaflet of the plasma membrane. This concept has now been tested in living cells using an assay sensitive to the lateral distribution of proteins in membranes over sub-micron distances.     

Aggregation, lipid exchange, and metastable phases of dimyristoylphosphatidylethanolamine vesicles

A new fluorescent lipid analogue, bimanephosphatidylcholine, has been synthesized for use in lipid bilayers. This probe is well suited as an energy-transfer donor with N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine as the acceptor. Dimyristoylphosphatidylethanolamine vesicles are prepared by sonication at pH 9 and characterized by electron microscopy and other methods. Resonance energy transfer between separately labeled donor and acceptor vesicles is monitored during HCl-induced aggregation to determine the kinetics of lipid randomization. Light scattering is also monitored to measure the kinetics of aggregation. The light scattering shows a marked reversal with NaOH while the energy transfer does not, indicating lipid exchange during a reversibly aggregated state; the extent of energy transfer suggests that only lipids in the outer monolayers exchange. The gel to liquid-crystalline phase transition temperature in HCl-treated vesicles is found to be 47 degrees C with diphenylhexatriene. The initial sonicated dispersion does not show a sharp phase transition. In vesicles labeled with both donor and acceptor probes, a small, irreversible increase in energy transfer is obtained upon lowering and then restoring the pH. These results suggest a metastable phase in the sonicated vesicles containing a randomized distribution of lipid and probes within the bilayers; the thermodynamically favored phase, whose formation is triggered by the pH shock, contains domains within which the probe lipids are more highly concentrated.     

Immobilized carbonic anhydrase: preparation,characteristics and biotechnological applications

Carbonic anhydrase (CA) is an essential metalloenzyme in living systems for accelerating the hydration and dehydration of carbon dioxide. CA-catalyzed reactions can be applied in vitro for capturing industrially emitted gaseous carbon dioxide in aqueous solutions. To facilitate this type of practical application, the immobilization of CA on or inside solid or soft support materials is of great importance because the immobilization of enzymes in general offers the opportunity for enzyme recycling or long-term use in bioreactors. Moreover, the thermal/storage stability and reactivity of immobilized CA can be modulated through the physicochemical nature and structural characteristics of the support material used. This review focuses on (i) immobilization methods which have been applied so far, (ii) some of the characteristic features of immobilized forms of CA, and (iii) biotechnological applications of immobilized CA. The applications described not only include the CA-assisted capturing and sequestration of carbon dioxide, but also the CA-supported bioelectrochemical conversion of CO₂ into organic molecules, and the detection of clinically important CA inhibitors. Furthermore, immobilized CA can be used in biomimetic materials synthesis involving cascade reactions, e.g. for bone regeneration based on calcium carbonate formation from urea with two consecutive reactions catalyzed by urease and CA.     

Preparation and characteristics of lipid vesicles

Summary As determined by electron microscopy, lipid sonicated in buffer initially forms large vesicles which may be multilamellar. Prolonged sonication results in a population of vesicles of smaller, but not uniform diameters. These vesicles are bounded by only one bilayer. The lipid suspension can be partially fractionated according to size by column chromatography. A fraction of the eluate has been selected for further study. The weight-average vesicle weight and average radius of gyration are obtained by lightscattering measurements. The volume of buffer enclosed by the vesicles is determined using¹⁴C- or³H-labelled sugars as a marker. These values are in reasonable agreement with the corresponding values calculated from the size distribution of the vesicle fraction obtained by electron microscopy.     

Microbial production of short and medium chain esters: Enzymes,pathways, and applications

Sustainable production of bulk chemicals is one of the major challenges in the chemical industry, particularly due to their low market prices. This includes short and medium chain esters, which are used in a wide range of applications, for example fragrance compounds, solvents, lubricants or biofuels. However, these esters are produced mainly through unsustainable, energy intensive processes. Microbial conversion of biomass-derived sugars into esters may provide a sustainable alternative. This review provides a broad overview of natural ester production by microorganisms. The underlying ester-forming enzymatic mechanisms are discussed and compared, with particular focus on alcohol acyltransferases (AATs). This large and versatile group of enzymes condense an alcohol and an acyl-CoA to form esters. Natural production of esters typically cannot compete with existing petrochemical processes. Much effort has therefore been invested in improving in vivo ester production through metabolic engineering. Identification of suitable AATs and efficient alcohol and acyl-CoA supply are critical to the success of such strategies and are reviewed in detail. The review also focusses on the physical properties of short and medium chain esters, which may simplify downstream processing, while limiting the effects of product toxicity. Furthermore, the esters could serve as intermediates for the synthesis of other compounds, such as alcohols, acids or diols. Finally, the perspectives and major challenges of microorganism-derived ester synthesis are presented.     

Charged lipid vesicles: effects of salts on bending rigidity, stability, and size

The swelling behavior of charged phospholipids in pure water is completely different from that of neutral or isoelectric phospholipids. It was therefore suggested in the past that, instead of multilamellar phases, vesicles represent the stable structures of charged lipids in excess water. In this article, we show that this might indeed be the case for dioleoylphosphatidylglycerol and even for dioleoylphosphatidylcholine in certain salts. The size of the vesicles formed by these lipids depends on the phospholipid concentration in a way that has been predicted in the literature for vesicles of which the curvature energy is compensated for by translational entropy and a renormalization of the bending moduli (entropic stabilization). Self-consistent field calculations on charged bilayers show that the mean bending modulus kc and the Gaussian bending modulus k have opposite sign and /k/>kc, especially at low ionic strength. This has the implication that the energy needed to curve the bilayer into a closed vesicle Eves=4pi(2kc+k) is much less than one would expect based on the value of kc alone. As a result, Eves can relatively easily be entropically compensated. The radii of vesicles that are stabilized by entropy are expected to depend on the membrane persistence length and thus on kc. Experiments in which the vesicle size is studied as a function of the salt and the salt concentration correlate well with self-consistent field predictions of kc as a function of ionic strength.     

Bacterial outer membrane vesicles: New insights and applications

Outer membrane vesicles (OMVs) (～50–250?nm in diameter) are produced by both pathogenic and nonpathogenic bacteria as a canonical end product of secretion. In this review, we focus on the OMVs produced by gram-negative bacteria. We provide an overview of the OMV structure, various factors regulating their production, and their role in modulating host immune response using a few representative examples. In light of the importance of the diverse cargoes carried by OMVs, we discuss the different modes of their entry into the host cell and advances in the high-throughput detection of these OMVs. A conspicuous application of OMVs lies in the field of vaccination; we discuss its success in immunization against human diseases such as pertussis, meningitis, shigellosis and aqua-farming endangering diseases like edwardsiellosis.     

VESICA: Computer graphics modeling of lipid vesicles

A vesicle simulation and computer analysis program, VESICA, is described which employs spherical projections of triangularly tessellated icosahedra to produce molecular graphics models of the three-dimensional structures of lipid vesicles. The program is used to analyze the molecular architecture of small unilamellar vesicles of dipalmitoylphosphatidylcholine and is demonstrated as a worthwhile investigative tool for determining the factors that govern the minimum vesicle size.     

Novel multilayered lipid vesicles: comparison of physical characteristics of multilamellar liposomes and stable plurilamellar vesicles

The preparation of a new kind of multilayered liposome, called a stable plurilamellar vesicle (SPLV), is described. Although SPLVs and classical multilamellar vesicles (MLVs) are made of the same materials and appear overtly similar in the electron microscope, the two types of vesicles differ as determined by stability, entrapment efficiency, electron spin resonance (ESR), NMR, X-ray diffraction, and biological effects. It is demonstrated that, contrary to what has been assumed, classical MLVs exclude solutes during their formation and, thus, are under a state of osmotic compression. By contrast, the SPLV process produces liposomes that are not compressed. The effects of osmotic compression are discussed. It is suggested that the state of osmotic stress is an important variable that distinguishes various types of liposomes and that has significant physical and biological consequences.     

Entrapment of lipid vesicles and membrane protein-lipid vesicles in gel bead pores

Phospholipid vesicles were entrapped in gel beads of Sepharose 6B and Sephacryl S-1000 during vesicle preparation by dialysis. Egg-yolk phospholipids solubilized with cholate or octyl glucoside were dialysed together with gel beads for 2.5 days in a flat dialysis bag. Some vesicles were formed in gel bead pores and vesicles of sufficient size became trapped. Red cell membrane protein-phospholipid vesicles could be immobilized in the same way. Non-trapped vesicles were carefully removed by chromatographic procedures and by centrifugation. The amount of entrapped vesicles increased with the initial lipid concentration and was dependent on the relative sizes of vesicles and gel pores. The largest amount of trapped vesicles, corresponding to 9.5 mumol of phospholipids per ml gel, was achieved when Sepharose 6B gel beads were dialysed with cholate-solubilized lipids at a concentration of 50 mM. In this case the vesicles had an average diameter of 60 nm and an internal volume of 15 microliters/ml gel. The amount of vesicles trapped in Sephacryl S-1000 gel beads upon dialysis under the same conditions was smaller: 2.2 mumol of phospholipids per ml gel. Probably most of the gel pores were too large to trap such vesicles. Larger vesicles, with an average diameter of 230 nm, were entrapped in the Sephacryl S-1000 matrix in an amount corresponding to 3.0 mumol phospholipids per ml gel upon dialysis of the gel beads and octyl glucoside-solubilized lipids at a concentration of 20 mM. The internal volume of these vesicles was 22 microliters/ml gel. The yield of immobilized phospholipids was up to 19%. The entrapped vesicles were somewhat unstable: 9% of the phospholipids were released during 9 days of storage at 4 degrees C. By the dialysis entrapment method vesicles can be immobilized in the gel beads without using hydrophobic ligands or covalent coupling.     

Purified curdlan and its hydroxyalkyl derivatives: preparation, properties and applications

Curdlan, a β-1,3-glucan produced by fermentation of Alkaligenes faecalis, is a non-ionic gel-forming polysaccharide that is, along with its hydroxalkyl derivatives, a potentially important matrix for life science applications. The commercially available material contains residual nucleic acids, cellular debris and other contaminants that can interfere with electrophoretic separations and visualization procedures. Simple procedures have been developed for purification of the curdlan and coherent gel formation. Curdlan gels can be formed in a variety of chaotrope-containing, or chaotropic, solvents, including 40% formamide, 7 M urea. The chaotropes can be retained or subsequently removed by leaching. Heat treatment before or after leaching enables thermostable gel formation. Hydroxyethyl and glyceryl derivatives of curdlan have been prepared. Depending on the degree of substitution (DS), a whole spectrum of derivatives with a range of unique properties can be obtained. Lower DS hydroxyethyl derivatives form clear, elastic gels in 6 M urea, while the higher DS hydroxyethyl derivatives are soluble in hot water and gel on cooling to form clear, elastic, thermoreversible gels. Partial depolymerization of curdlan by γ-irradiation reduces the viscosity of subsequent preparations, enabling the preparation of higher concentration, more sieving gels. Use of selected preparations to form unique matrices for electrophoretic separations has been demonstrated.