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.     

Hydrolysis of phosphatidylcholine in phosphatidylcholine-cholate mixtures by porcine pancreatic phospholipase A2

Pancreatic phospholipase A2 (PLA2)-catalyzed hydrolysis of egg yolk phosphatidylcholine (PC) in mixed PC-cholate systems depends upon composition, structure, and size of the mixed aggregates. The hydrolysis of PC-cholate-mixed micelles made of an equal number of PC and cholate molecules is consistent with a Km of about 1 mM and a turnover number of about 120 s-1. Increasing the cholate/PC ratio in the micelles results in a decreased initial velocity. Hydrolysis of cholate-containing unilamellar vesicles is very sensitive to the ratio of cholate to PC in the vesicles. The hydrolysis of vesicles with an effective cholate/PC ratio greater than 0.27 is similar to that of the mixed micelles. The time course of hydrolysis of vesicles with lower effective ratios is similar to that exhibited by pure dipalmitoyl-phosphatidylcholine (DPPC) large unilamellar vesicles in the thermotropic phase transition region. In the latter two cases, the rate of hydrolysis increases with time until substrate depletion becomes significant. The reaction can be divided phenomenologically into two phases: a latency phase where the amount of product formed is a square function of time (P(t) = At2) and a phase distinguished by a sudden increase in activity. The parameter A, which describes the activation rate of the enzyme during the initial phase in a quantitative fashion, increases with increasing [PLA2], decreasing [PC], decreasing vesicle size, and increasing relative cholate content of the vesicles. The effect of [PLA2] and [PC] on the hydrolysis reaction is similar to that found with pure DPPC unilamellar vesicles in their thermotropic phase transition region. The effect of cholate on the hydrolysis reaction is similar to that of temperature variation within the phase transition of temperature variation within the phase transition of DPPC. These results are consistent with our previously proposed model, which postulates that activation of PLA2 involves dimerization of the enzyme on the substrate surface and that the rate of activation is directly proportional to the magnitude of lipid structural fluctuations. It is suggested that large structural fluctuations, which exist in the pure lipid system in the phase transition range, are introduced into liquid crystalline vesicles by the presence of cholate and thus promote activation of the enzyme.     

Self-replicating micelles — A chemical version of a minimal autopoietic system

Reverse micelles hosting the internal production of the surfactant are proposed as experimentally feasible models of simple (or minimal) autopoietic systems. We describe the conditions under which these may be formed and their possible biological implications. The micellar systems considered here turn out also to exhibit a capacity for self-reproduction through fragmentation under plausible conditions, thus constituting also a minimal experimental model for prebiotic self-reproduction.     

Micelle-vesicle transition of egg phosphatidylcholine and octyl glucoside

The dissolution and formation of egg phosphatidylcholine (PC) vesicles by the detergent octyl glucoside were examined systematically by using resonance energy transfer between fluorescent lipid probes, turbidity, and gel filtration chromatography. Resonance energy transfer was exquisitely sensitive to the intermolecular distance when the lipids were in the lamellar phase and to the transitions leading to mixed micelles. Turbidity measurements provided information about the aggregation of lipid and detergent. Several reversible discrete transitions between states of the PC-octyl glucoside system were observed by both methods during dissolution and vesicle formation. These states could be described as a series of equilibrium structures that took the forms of vesicles, open lamellar sheets, and mixed micelles. As detergent was added to an aqueous suspension of vesicles, the octyl glucoside partitioned into the vesicles with a partition coefficient of 63. This was accompanied by leakage of small molecules and vesicle swelling until the mole fraction of detergent in the vesicles was just under 50% (detergent:lipid ratio of 1:1). Near this point, a transition was observed by an increase in turbidity and release of large molecules like inulin, consistent with the opening of vesicles. Both a turbidity maximum and a sharp increase in fluorescence were observed at a detergent to lipid mole ratio of 2.1:1. This was interpreted as the lower boundary of a region where both lamellar sheets and micelles are at equilibrium. At a detergent:lipid ratio of 3.0:1, another sharp change in resonance energy transfer and clarification of the suspension were observed, demarcating the upper boundary of this two-phase region. This latter transition is commonly referred to as solubilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aggregation Properties of Biological Amphiphiles: Micelles and Vesicles

Abstract

Selfaggregation of amphiphilic molecules in aqueous solutions is discussed in terms of their geometrical properties. Gangliosides, sialic-acid containing glycosphingolipids, are an interesting example of biological amphiphiles which can selfaggregate into different shapes ranging from vesicles to micelles, depending on the relative extension and conformation of their saccharidic headgroups. The remarkable differences in the mechanical properties of ganglioside and lecitine vesicles are also discussed by means of geometrical considerations.     

Question 7: New Aspects of Interactions Among Vesicles

In this short article I discuss the relevance of two aspects of vesicle reactivity that are germane to understand the role of compartments in the origin of early cells. Studies of vesicle self-reproduction indicate that simple vesicles can grow and divide, maintaining inside most of their content and giving rise to a simple autopoietic system. New aspects of vesicle reactivity are also introduced, such as selection and competition processes within vesicle populations, emphasizing the concepts of vesicle diversity, inter-vesicles and vesicles–environment interactions, intended as synthetic analogs of primitive ‘ecological’ processes. Presented at: International School of Complexity – 4th Course: Basic Questions on the Origins of Life; “Ettore Majorana” Foundation and Centre for Scientific Culture, Erice, Italy, 1–6 October 2006.     

Kinetics of ganglioside transfer between liposomal and synaptosomal membranes

The transfer of ganglioside GM1 from micelles to membranes and between different membrane populations has been examined by using a pyrene fatty acid derivative of the ganglioside. The transfer of gangliosides from micelles to membranes depends on the physical state as well as the molecular composition of the acceptor vesicles. At 30 degrees C, the transfer of micellar gangliosides to dipalmitoylphosphatidylcholine (DPPC) large unilameller vesicles (Tm = 41.3 degrees C) is characterized by a rate constant of 0.01 min-1; at 48 degrees C, however, the rate constant is 0.11 min-1. Below the phase transition temperature, the activation energy is 25 kcal/mol whereas above the phase transition it is 17 kcal/mol. Similar experiments performed with synaptic plasma membranes yielded a rate constant of 0.05 min-1 at 37 degrees C. The rate of transfer of ganglioside molecules, asymmetrically located on the outer layer of donor vesicles, to acceptor vesicles lacking ganglioside depends on the physical state of both the donor and acceptor vesicles. For the transfer of ganglioside from DPPC (donor) vesicles to dimyristoylphosphatidylcholine (DMPC) (acceptor) vesicles, the rates were essentially zero at 15 degrees C in which both vesicle populations were in the gel phase, 0.008 min-1 at 30 degrees C in which DPPC is in the gel phase and DMPC is in the fluid phase, and 0.031 min-1 at 48 degrees C in which both vesicle populations are in the fluid phase. The transfer of ganglioside from DPPC vesicles to synaptic plasma membranes was also dependent on the physical state of the donor vesicles and showed an inflection point at the phase transition temperature of DPPC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydrolysis of dipalmitoylphosphatidylcholine large unilamellar vesicles by porcine pancreatic phospholipase A2

The interaction between dipalmitoylphosphatidylcholine large unilamellar vesicles and porcine pancreatic phospholipase A2 has been studied under a variety of conditions. It was found that the presence of large unilamellar vesicles inhibits the hydrolysis of small unilamellar vesicles at room temperature, and reaction calorimetric experiments showed that protein-lipid interactions in the absence of Ca2+ occur in the gel state with a stoichiometry of about 40 phospho-lipid molecules/protein-binding site. However, hydrolysis can be induced in the gel state under conditions of osmotic shock. On the other hand, hydrolysis is usually observed within the lipid transition temperature range, but then it occurs only after a latency phase during which the hydrolysis is very slow. The duration of this latency phase reaches a minimum near the phase transition temperature. However, if the enzyme-substrate mixture is heated from low temperatures (continuously or by a temperature jump) to a temperature within the phase transition region, hydrolysis occurs instantaneously. These results are in accordance with the conclusions of the preceding paper (Menashe, M., Romero, G., Biltonen, R. L., and Lichtenberg, D. (1986) J. Biol. Chem. 261, 5328-5333) that effective binding of the enzyme to lipid vesicles occurs relatively rapidly in the gel state and that activation of the enzyme-substrate complex requires the existence of structural irregularities in the lipid bilayer. Although hydrolysis products may have a pronounced effect on the time course of the reaction in the transition range, instantaneous hydrolysis can be induced in the phase transition region in the absence of reaction products by appropriate manipulation of the experimental conditions during which no reaction products are produced. Thus reaction products are not essential for activation of porcine pancreatic phospholipase A2. Furthermore, it is shown that the fraction of lipid hydrolyzed during the latency period is a function of the initial substrate concentration in a manner inconsistent with the proposition that the accumulation of a constant critical fraction of reaction products is the basis for activation. Comparison of the results of this study with those of the preceding paper strongly support the previously proposed reaction scheme.     

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.     

Efficient kinetic resolution of dl-menthol by lipase-catalyzed enantioselective esterification with acid anhydride in fed-batch reactor

Acid anhydrides were used as highly reactive and non-water-producing acyl donors for hydrolase-catalyzed enantioselective esterification. Efficient kinetic resolution of dl-menthol has been achieved via lipase-catalyzed enantioselective esterification in cyclohexane when propionic anhydride as an acyl donor was continuously fed into a reactor containing dl-menthol and Candida cylindracea lipase OF 360, while a high concentration of the acid anhydride in a batch reaction system with a dehydrated organic solvent did not facilitate the reaction, because water necessary for the enzyme function was consumed by the competing hydrolysis of the anhydride catalyzed by the same enzyme. The efficiency of this fed-batch reaction system using acid anhydride was higher and the enzyme stability in repeated use was much better than those of conventional batch and fed-batch reaction systems using propionic acid as an acyl donor. The optical purity (more than 98% e.e.) of the l-menthyl ester produced in the fed-batch system using the anhydride was comparable to that in the system using the corresponding acid. *** DIRECT SUPPORT *** AG903062 00002     

Kinetics of interfacial catalysis by phospholipase A2 in intravesicle scooting mode,and heterofusion of anionic and zwitterionic vesicles

In this and the following three papers we examine the kinetics of action of pig pancreatic phospholipase A₂ on vesicles of anionic phospholipids without any additives. The results provide the first unequivocal demonstration of interfacial catalysis in intravesicle scooting mode. In this paper we describe the conditions in which the action of pig pancreatic phospholipase A₂ on DMPMe (ester) vesicles in the absence of any additive commences without a latency. Under these conditions the free monomer substrate concentration is insignificant; the bilayer enclosed vesicle organization remains intact even when all the substrate in the outer monolayer has been hydrolyzed; the rate of intervesicle exchange and the rate of transbilayer movement (flip-flop) of molecules is negligibly slow; and the rate of fusion of vesicles is insignificant. Thus an enzyme molecule bound to one vesicle hydrolyzes all the DMPMe molecules in the outer monolayer of the vesicle by a first-order process with a rate constant of 0.6 per min at 30°C; or viewed another way, one enzyme molecule in a DMPMe vesicle can hydrolyze all the available substrate molecules at the rate of 3000 per min. At low anion concentrations excess substrate vesicles are not hydrolyzed unless the rate of intervesicle exchange of the bound enzyme is stimulated by anions in the aqueous phase. Higher calcium concentrations promote not only homofusion of DMPMe vesicles but also heterofusion of DMPMe and DMPC vesicles. It is proposed that calcium-induced isothermal lateral phase separation in DMPMe vesicles induces defects in the bilayer organization, and such defects are the sites for phospholipase A₂ binding and for heterofusion with DMPC (ester) vesicles which do not have such sites.     

Hydrolysis of di- and trisialo gangliosides in micellar and liposomal dispersion by bacterial neuraminidases

The hydrolysis of di- and trisialo gangliosides by bacterial neuraminidases was investigated. Slow rates of hydrolysis were obtained with micellar dispersions of the pure gangliosides; the rates increased considerably with mixtures of ganglioside and phospholipids, such as phosphatidylcholine or sphingomyelin. The greatest rates of hydrolysis were obtained with mixtures containing 5-10 mol% ganglioside and 90-95% phospholipid. With the aid of the nonpenetrating reagent trinitrobenzenesulfonic acid, it was ascertained that this mixture consisted of sealed, unilamellar vesicles in which the ganglioside was distributed symmetrically between the two layers of the liposome. When the relative proportion of the ganglioside was increased, the dispersions contained liposomes admixed with micelles of ganglioside and phospholipid. The rates of hydrolysis of the ganglioside could be correlated with the percentage of sealed vesicles in each mixture. Experiments in which another ganglioside (GM1) or cholesterol was incorporated into the mixed dispersions further supported this conclusion. It is suggested that the rate of hydrolysis is affected predominantly by interactions between the carbohydrate chains of ganglioside molecules. The data emphasize that ganglioside metabolism can be best studied when the latter are part of biological or model membranes.     

Two-Step biocatalytic conversion of an ester to an aldehyde in reverse micelles

Lipases from Candida cyclindracea (L-1754) and wheat germ (L-3001) have been used to hydrolyze esters to their corresponding alcohols and acids in reverse micelles. Alcohol dehydrogenase from baker's yeast (YADH) was subsequently used to reduce the alcohol products to aldehydes. Cofactor recycling in the redox reaction was achieved using a sacrificial cosubstrate, as described previously. Four surfactants (sodium dioctylsulfosuccinate, Nonidet P-40 with Triton X-35, polyoxyethylene, 10-cetyl-ether, polyoxyethylene sorbitan trioleate) were employed to determine the effect of amphiphile on ester hydrolysis and redox reaction rates separately. The effect of type of organic solvent, W(0) [(water]/[surfactant)], and substrate concentration on separte enzyme activity were also investigated. A brief investigation of a single phase, two-step reaction catalyzed by the combination of lipase and YADH in reverse micelles is also reported. The activities of the enzymes are significantly different when used together instead of independently. (c) 1994 John Wiley & Sons, Inc.     

Studies on the chemical stability of propylene glycol alginate esters

The chemical stability of propylene glycol alginates (PGAs) has been examined. Under acidic conditions the ester groups in PGA are stable to hydrolysis but hydrolytic degradation of the glycosidic linkages in the polysaccharide backbone occurs. Under alkaline conditions the ester groups are hydrolysed with the primary 2-hydroxyprop-1-yl ester groups being more susceptible than secondary 1-hydroxyprop-2-yl ester groups, with little degradation of the polysaccharide backbone. Sodium carbonate-bicarbonate buffer was a much more effective hydrolysing reagent than sodium hydroxide at the same concentration and pH, and the rate of hydrolysis was greatly accelerated by increasing the hydrolysis temperature. Acetate, citrate and phosphate ions accelerated the rate of hydrolysis of the ester groups in PGA when added to the sodium hydroxide hydrolysing reagent. Hydrolysis of the ester groups in PGA with sodium hydroxide was unaffected by the addition of imidazole. However hydrolysis of the ester groups in PGA with sodium hydroxide in the presence of 1-aminobutane led to the formation of an alginate amide in which only the primary 2-hydroxylprop-1-yl ester groups were present, suggesting that a nucleophilic substitution of primary ester groups by amine groups is involved in the reaction.     

Internal lipid synthesis and vesicle growth as a step toward self-reproduction of the minimal cell

One of the major properties of the semi-synthetic minimal cell, as a model for early living cells, is the ability to self-reproduce itself, and the reproduction of the boundary layer or vesicle compartment is part of this process. A minimal bio-molecular mechanism based on the activity of one single enzyme, the FAS-B (Fatty Acid Synthase) Type I enzyme from Brevibacterium ammoniagenes, is encapsulated in 1-palmitoyl-2oleoyl-sn-glycero-3-phosphatidylcholine (POPC) liposomes to control lipid synthesis. Consequently molecules of palmitic acid released from the FAS catalysis, within the internal lumen, move toward the membrane compartment and become incorporated into the phospholipid bilayer. As a result the vesicle membranes change in lipid composition and liposome growth can be monitored. Here we report the first experiments showing vesicles growth by catalysis of one enzyme only that produces cell boundary from within. This is the prototype of the simplest autopoietic minimal cell.     

States of aggregation and phase transformations in mixtures of phosphatidylcholine and octyl glucoside

The result of mixing varying concentrations of the nonionic detergent octyl glucoside (OG) with small unilamellar vesicles (SUV) of egg phosphatidylcholine (PC) made by sonication depends on the ratio between OG and PC in the mixed aggregates. When this molar ratio (Re) is lower than 1.4, the detergent partitions between the PC vesicles and the aqueous medium with a partition coefficient of K = 0.033 mM-1. As a consequence of introduction of OG into the bilayers, the vesicles grow in size. The resultant vesicles have a mean diameter that is an increasing function of Re and is independent of the total PC concentration. Experiments in which the vesicles were loaded with high molecular weight dextran prior to being exposed to OG suggest that the mechanism responsible for the size growth involves lipid transfer rather than fusion. Mixtures with Re values within the range of 1.4-3.2 separate into two macroscopic phases: The lower phase is clear but very viscous. It contains constant OG and PC concentrations and is characterized by an Re value of 3.2, independent of the composition of the whole dispersion. The upper phase contains vesicles of varying concentrations of OG and PC, but a constant Re of 1.4. When the saturating level of 1.4 OG molecules per PC molecule is approached, the concentration of OG monomers in the aqueous medium reaches the value of 16.6 +/- 0.3 mM, which is the apparent cmc of OG in the lipid-containing medium. OG-PC mixed micelles contain at least 3.2 OG molecules per PC molecule. The mixed micelles present at Re = 3.2 apparently have the shape of oblate ellipsoids with a minor axis of about 2 nm and two major axes of about 25 nm. The surface area of the mixed micelles at this point is just sufficient for them to undergo conversion into the smallest possible spherical vesicles of a radius of 12 nm. At Re values above 3.2, the major axis of the mixed micelles becomes smaller as Re increases, while at values of Re below 3.2 the micelles would have been expected to grow very rapidly with decreasing Re. This may explain the partial vesicle closure occurring below Re = 3.2.     

Synergetics of the Membrane Self-Assembly: A Micelle-to-Vesicle Transition

A model approach is developed to study intermediate steps and transientstructures in a course of the membrane self-assembly. The approach isbased on investigation of mixed lipid/protein-detergent systems capable ofthe temperature induced transformation from a solubilized micellar stateto closed membrane vesicles. We performed a theoretical analysis ofself-assembling molecular structures formed in binary mixtures ofdimyristoylphosphatidylcholine (DMPC) and sodium cholate (NaC). Thetheoretical model is based on the Helfrich theory of curvature elasticity,which relates geometrical shapes of the structures to their free energy inthe Ginzburg-Landau approximation. The driving force for the shapetransformation is spontaneous curvature of amphiphilic aggregates which isnonlinearly dependent on the lipid/detergent composition. An analysis ofthe free energy in the regular solution approximation shows that theformation of mixed structures of different shapes (discoidal micelles,rod-like micelles, multilayer membrane structures and vesicles) ispossible in a certain range of detergent/lipid ratios. A transition fromthe flat discoidal micelles to the rod-like cylindrical micelles isinduced by curvature instabilities resulting from acyl chain melting andinsertion of detergent molecules into the lipid phase. Nonideal mixing ofthe NaC and DMPC molecules results in formation of nonideal cylindricalaggregates with elliptical cross section. Further dissolution of NaCmolecules in DMPC may be accompanied with a change of their orientation inthe lipid phase and leads to temperature-induced curvature instabilitiesin the highly curved cylindrical geometry. As a result the rod-likemicelles fuse into less curved bilayer structures which transformeventually to the unilamellar and multilamellar membrane vesicles. Thetheoretical analysis performed shows that a sequence of shapetransformations in the DMPC/NaC mixed systems is determined by thesynergism of four major factors: detergent/lipid ratio, temperature (acylchain melting), DMPC and NaC mixing, and reorientation of NaC molecules inmixed aggregates.     

Structure of molecular aggregates of 1-(3-sn-phosphatidyl)-L-myo-inositol 3,4-bis(phosphate) in water

The molecular organization of 1-(3-sn-phosphatidyl)-L-myo-inositol 3,4-bis-(phosphate)/water systems is investigated over a wide range of lipid concentrations using X-ray diffraction, calorimetry, analytical ultracentrifugation, densitometry and viscometry. At high lipid concentrations, the lipid molecules are found to form a lamellar phase. The repeat distance increases from 60 to 120 A with increasing water content to 70 wt% and the surface area per lipid molecule increases from 41.7 A2 to a limiting value of 100 A2. On the other hand, at very low lipid concentrations the molecules are found to form not vesicles but micelles, the total molecular weight of which takes a value of 93,000. This finding revises the prevalent view that lipids containing two (or more) hydrocarbon chains form extended bilayers or vesicles, whereas single chained lipids form micelles.     

Comparative action of lysophospholipases on acyl-oxyester and acyl-thioester substrates in micellar and membrane-bound form

Phospholipid analogs in which the acyl-oxyester bond is replaced by an acyl-thioester bond represent convenient substrates for sensitive assays of lipolytic enzymes. It has previously been found that such thioester substrates are hydrolyzed at higher rates than their oxyester counterparts. For bovine liver lysophospholipase II the preferential hydrolysis of thioesters appeared to be due to the thioester linkage per se rather than to the formation of preferred interfaces. The preferential hydrolysis of thioesters persisted when thioester and oxyester substrates were presented to the enzyme either as mixed micelles or incorporated in the bilayer of phospolipid vesicles. The transbilayer distribution of thioester and oxyester substrates in sonicated phospholipid vesicles is identical with no apparent indications for transbilayer movement of both substrates.     

A new chemical procedure for the preparation of gangliosides carrying fluorescent or paramagnetic probes on the lipid moiety

A new chemical procedure is described for preparing labelled GM1 molecular species, carrying as acyl moiety pyrene-decanoic acid, 5-doxyl-stearic acid and 16-doxyl-stearic acid. It makes use of a mixed anhydride formed by ethylchloroformate and the labelled acyl chain, as the reagent for N-acylation of a deacetylated, deacylated GM1 ganglioside, which is prepared by alkaline hydrolysis of natural GM1. The reaction performed with a unitary GM1 derivative/mixed anhydride molar ratio, occurs with a yield of above 40%. The labelled deacetylated GM1 molecular species are then N-acetylated by means of acetic anhydride with quantitative yield. The chemical process of insertion of labelled fatty acid and reconstitution of GM1 ganglioside has been confirmed by GLC-MS and NMR analyses. Fluorescence and electron spin resonance experiments indicate that the labelled gangliosides behave similarly to natural GM1, in both the aggregation properties and the capability to be transferred from micelles to vesicular dispersions of phospholipids.