Short-chain Lecithins Research Articles

Spontaneous transmembrane insertion of membrane proteins into lipid vesicles facilitated by short-chain lecithins.

Functional reconstitution of the membrane protein bacteriorhodopsin into lipid vesicles is achieved by mixing aqueous suspensions of long-chain lecithins and purple membrane with the short-chain lecithin diheptanoylphosphatidylcholine (20 mol % of total lipid). The membrane protein is transmembranously inserted in the lipid bilayer of the vesicle and highly active as a light-energized proton pump. This rapid, easy, and gentle procedure might allow functional reconstitution of other membrane systems and isolated membrane proteins as well.

Hydrolysis of short-chain phosphatidylcholines by bee venom phospholipase A 2

In order to find out the aggregation state of the substrate, preferred by bee venom phospholipase A 2 (EC 3.1.1.4), its action on short-chain phosphatidylcholines with two identical (C 6 – C 10) fatty acids has been tested. The rate of hydrolysis as a function of acyl chain length showed a maximum at dioctanoylphosphatidylcholine. The effects of alcohols, NaCl and Triton X-100, which affect the aggregation state of phospholipids in water, were also studied. The addition of n-alcohol led to a significant inhibition of the hydrolysis of the substrates present in micellar form and activated the hydrolysis of substrates which form liposomes. The inhibitory effect increased with increasing length of the aliphatic carbon chain of the alcohol. Triton X-100 at low Triton/phospholipid molar ratios enhanced enzyme activity. These results do not agree with the accepted idea that bee venom phospholipase A 2 hydrolyzes short-chain lecithins in their molecularly dispersed form and that micelles cannot act as substrates. The data indicate that short-chain lecithins in the aggregated state are hydrolyzed and that the requirements of bee venom phospholipase A 2 for the aggregation state of the substrate are not strict.

Methyl branching in short-chain lecithins: are both chains important for effective phospholipase A2 activity?

Several seven-carbon fatty acyl lecithins with varied acyl chain branching have been synthesized and characterized as potential phospholipase A2 substrates. Micellar bis(4,4-dimethylpentanoyl) phosphatidylcholine, bis(5-methylhexanoyl)phosphatidylcholine, bis(3-methylhexanoyl)phosphatidylcholine, and bis(2-methylhexanoyl)phosphatidylcholine are poor substrates for phospholipase A2 (Naja naja naja). These branched lecithins also inhibit the hydrolysis of diheptanoylphosphatidylcholine by the enzyme with Ki values comparable to or smaller than the apparent Km of the linear compound. The terminally branched lecithins are excellent substrates for another surface-active hydrolytic enzyme, phospholipase C from Bacillus cereus. When only one acyl chain bears a methyl group, the hybrid lecithins 1-heptanoyl-2-(2-methylhexanoyl)phosphatidylcholine and 1-(3-methylhexanoyl)-2-heptanoylphosphatidylcholine are substrates comparable to diheptanoylphosphatidylcholine. Analysis of micellar structure and dynamics by 1H and 13C NMR spectroscopy, quasi-elastic light scattering, and comparison of critical micellar concentrations indicates little significant difference in the conformation and dynamics of these seven-carbon fatty acyl lecithin micelles, even when the methyl groups are adjacent to the carbonyls. Phospholipase A2 UV difference spectra induced by phospholipid binding imply different enzyme conformations or aggregation states caused by linear-chain and asymmetric-chain lipids compared to bis(methylhexanoyl)phosphatidylcholines. The differences in hydrolytic activity of phospholipase A2 against the branched-chain micellar lecithins can then be attributed to an enzyme-lipid interaction at the active site. The species with both fatty acyl chains branched bind to phospholipase A2 but are not turned over rapidly. Since poor enzymatic activity only occurs for lecithins with both chains methylated, the interaction of both chains with the enzyme must be important for catalytic efficiency.

Kinetic studies on the reactivation of d-β-hydroxybutyrate dehydrogenase with mixtures of short-chain lecithins

d-β-hydroxybutyrate dehydrogenase, purified as soluble, lipid-free apoenzyme (inactive) from rat liver mitochondria can be reactivated by the short-chain dihexanoyl, diheptanoyl, and dioctanoyl lecithins at the monomeric state, upon formation of a reversible enzyme-lecithin complex. Previous studies with these lecithins suggested that reactivation of the apoenzyme requires the simultaneous occupation of two identical, noninteracting lecithin binding sites via a rapid equilibrium random mechanism. The short-chain lecithins exhibited similar reactivating capacities, differing only in their affinities towards the enzyme. In order to further test that model, the reactivation of the apoenzyme was studied when two or three short-chain lecithins were simultaneously present in the reaction medium. The initial velocities were measured either as a function of the concentration of one lecithin while the other(s) were kept constant, or as a function of the total phospholipid concentration with mixtures of different lecithins at a constant molar ratio. The pertinent equations were derived on the principles of multiple equilibria with identical, noninteracting sites able to be occupied by any of the different lecithins present in the reaction medium, with the doubly occupied enzyme as the only active species. In agreement with the above-proposed model, the results obtained indicates that the molar fraction of the doubly occupied (active) enzyme species can be calculated from equilibrium considerations and that the maximal attainable with the different short-chain lecithins are similar.

Effect of apolipoprotein C-II on the lipoprotein lipase-catalyzed hydrolysis of dihexanoyl- and diheptanoyl-phosphatidylcholine

The effect of apolipoprotein C-II (apoC-II) on the lipoprotein lipase (LpL)-catalyzed hydrolysis of phospholipids was studied using purified bovine milk LpL and dihexanoyl (diC 6) and diheptanoyl (diC 7) phosphatidylcholine. In contrast to porcine pancreatic phospholipase A 2, the LpL-catalyzed hydrolysis of these short-chain lecithins was not enhanced at substrate concentrations above the critical micelle concentration of the lipids. Furthermore, apoC-II had no effect on enzyme catalysis.

Reactivation of D-beta-hydroxybutyrate dehydrogenase with short-chain lecithins: stoichiometry and kinetic mechanism.

D-beta-Hydroxybutyrate dehydrogenase (BDH), purified as soluble, lipid-free apoenzyme (inactive) from either beef heart or rat liver mitochondria, can be reactivated by short-chain lecithins in the monomeric state. The enzyme was reactivated with dihexanoyl- [PC(6:0)], diheptanoyl- [PC(7:0)], and dioctanoyllecithins [PC(8:0)]. The titration curves of enzyme activity as a function of the phospholipid concentration are consistent with a model in which the enzyme contains two identical, noninteracting lecithin binding sites. The simultaneous occupation of these sites (via an equilibrium random mechanism) is required to activate the apoenzyme. Similar results were obtained with both rat liver and beef heart apoenzymes. The maximal velocities obtained with the different lecithins were similar [110-140 mumol of NAD+ reduced min-1 (mg of protein)-1]. The KL values (the apparent dissociation constants of the lecithin-site complexes) were 1.2 X 10(-4) M [PC(8:0)], 1.5 X 10(-3) M [PC(7:0)], and 4.5 X 10(-3) M [PC(6:0)] at 37 degrees C. This was confirmed by using phospholipase A2 to compete with the dehydrogenase for the lecithin monomers. Comparison of the delta G degrees values for complex formation with the different lecithins shows an average contribution of approximately 2.4 kJ/mol (0.9RT) per CH2 group. The interaction of the apolar moiety of lecithin with the protein seems to be essential for effective binding of phosphatidylcholine to apoBDH. The delta G degrees values, when combined with the estimated delta H degrees values, suggest that the binding of lecithin to the apoenzyme is approximately 60% enthalpy and approximately 40% entropy driven.

Amphiphile-mediated activation of soluble adenylate cyclase of Bordetella pertussis

The adenylate cyclase activity of Bordetella pertussis culture supernatants is activated 3- to 10-fold by various amphiphiles including many classes of phospholipids and nonionic detergents. Gangliosides are inhibitory. The stimulation affects the V max and not the K m . Neither the nature of the polar head group, the length of the fatty acid chains, nor the hydrophile-lipophile balance (in the Triton X series) are major determinants for activation. Short-chain lecithins activate as monomers, whereas long-chain lecithins activate only above the critical micelle concentrations, suggesting high-affinity hydrophobic binding sites. Judged by EGTA inhibition, the amphiphile-mediated activation requires Ca 2+ in the absence of calmodulin. In addition, amphiphiles sensitize the adenylate cyclase to Ca 2+/calmodulin and are also synergistic with calmodulin for maximal stimulation.

Using Short-Chain Lecithins to Study the Surface Behavior of Lipolytic Enzymes

Detailed mechanistic studies of lipases and phospholipases are hampered by poorly defined surfaces. In particular, manipulation of lipid component concentrations can drastically affect particle structure and/or enzymatic activity. Short-chain lecithin molecules are a particularly useful building block of complex but characterizable micellar particles. These lecithins, with four to eight carbons in the fatty acyl chains, exist as monomers in aqueous solution and form micelles above a critical micelle concentration (c.m.c.). Their detergent properties allow them to solubilize large fractions of lipophilic substances such as triglycerides (0.5 mol fraction) and cholesterol (0.18 mol fraction). We have used 13C NMR spectroscopy of these lecithins to (a) examine the conformation and motion of individual sn-I /sn-2 chains for monomeric and micellar lecithin; (b) assess the conformational effects of chemical changes in the lecithin molecule; and (c) characterize mixed particles consisting of short-chain lecithins, triglyceride and/or cholesterol that mimic lipoprotein structure. The characterized particles are then used in kinetic studies of lipolytic enzymes.

Accessibility of the active site of crotoxin B in the crotoxin complex

G. Canziani, C. Seki and J. C. Vidal. Accessibility of the active site of crotoxin B in the crotoxin complex. Toxicon 20, 809–822, 1982.—Basic phospholipases A and the crotoxin complex isolated from Crotalus durissus terrificus venom exhibited similar initial reaction rates, time course and degree of hydrolysis of synthetic short chain lecithins in the monomeric state. Although monomeric lecithins seem to promote dissociation of crotoxin up to a certain extent, this cannot explain the high activity observed with the complex. The crotoxin complex is able to bind the non-hydrolyzable analog d-diheptanoyllecithin, as demonstrated by equilibrium gel-filtration, with a dissociation constant of 0.12mM. This value is similar to the dissociation constant of the crotoxin B- d-diheptanoyllecithin complex (about 0.13mM), estimated from the protection against enzyme inactivation by p-bromophenacyl bromide, which further supports the free accessibility of the substrate to the enzyme active site in the crotoxin complex. The lack of enzyme inactivation when crotoxin is treated with p-bromophenacyl bromide may be interpreted in terms of the specific requirements of the reagent to react with the enzyme rather than protection of the active site. Crotoxin B inhibition by complex formation with crotoxin A, which is not apparent on monomeric substrates, seems not to involve the active site of the enzyme.

Cholesterol solubilization by short-chain lecithins: characterization of mixed micelles and cholesterol oxidase activity.

The synthetic short-chain lecithins diheptanoylphosphatidylcholine and dioctanoylphosphatidylcholine solubilize cholesterol up to 10 and 18 mol %, respectively. The half-time for diheptanoylphosphatidylcholine solubilization of solid cholesterol is 80 (+/- 30) min. This is much faster than Triton X-100 micelle or egg lecithin vesicle solubilization of solid cholesterol. Both the broadening of lecithin and [4-13C]cholesterol carbon resonances by Mn2+ and the observation of surface dilution kinetics for phospholipase A2 (Naja naja naja) and phospholipase C (Bacillus cereus) hydrolysis of the lecithins indicate that the cholesterol 3 beta-hydroxyl group resides at the particle surface exposed to solvent. Analysis of lecithin 13C chemical shifts suggests that cholesterol causes the short-chain lecithin acyl chains to become slightly more trans, although to a lesser extent than it affects egg lecithin chains in liposomes. Lecithin motion as characterized by 13C T1s and line widths is unaffected by the incorporation of cholesterol. [3,4-13C2]Cholesterol line widths are 5-10-fold narrower in these mixed micelles than in egg lecithin sonicated vesicles, while T1s in the two systems are comparable. These mixed micelles serve as substrates for cholesterol oxidase (Nocardia erythropolis) with a 40-fold rate increase over comparable cholesterol concentrations in egg lecithin vesicles. Part of this rate enhancement can be understood as an increase in interfacial area available to cholesterol oxidase in the micellar systems. These studies suggest that cholesterol oxidase has a weaker affinity for interfaces than other surface active enzymes.

Thermodynamics and kinetics of micellization in aqueous solutions of some spin-labeled short-chain lecithins

The micellization of three spin-labeled lecithins, 1-acyl-2[4′-( N-oxyl-4″,4″-dimethyloxazolidine)-pentanoyl]-sn-glycero-3-phosphorylcholine, was investigated by electron spin resonance (ESR) spectroscopy. Data for both the thermodynamics of micellization and the kinetics of the monomermicelle exchange are presented.

Carbon-13 nuclear magnetic resonance studies of short-chain lecithins. Motional and conformational characteristics of micellar and monomeric phospholipid

Carbon-13 nuclear magnetic resonance studies of short-chain lecithins. Motional and conformational characteristics of micellar and monomeric phospholipid

Regulation of phospholipase A2 activity by different lipid-water interfaces.

The activity of pancreatic phospholipase A2 (EC 3.1.1.4) is controlled not only by the architecture of the catalytic site, but is also strongly dependent on the penetrating power of the interface recognition site and the packing density of the lipid-water interface. The influence of the latter two factors on the interface activity has been investigated using chemically modified phospholipases A2 in which the NH2-terminal L-Ala8 has been replaced by DL-[3-13C]Ala, or in which the polypeptide chain has been elongated with DL-[3-13C]Ala. The [DL-(3-13C)Ala8]phospholipase A2 could be resolved into the pure diastereoisomers, [D-(3-13C)Ala8]phospholipase A2 and [L-(3-13C)Ala8]phospholipase A2 by elution on Sephadex G-100 in the presence of a micellar lipid-water interface, as well as by conventional ion exchange chromatography on carboxymethylcellulose. Similar procedures did not effect, however, a separation of DL-[3-13C]Ala7-phospholipase A2 into their respective diasteroisomers, indicating the strategic role of the NH2-terminal L-Ala8 residue in the interaction process between the enzyme and lipid-water interfaces. Kinetic experiments using various micellar short chain lecithins revealed the apparent absence of an interface recognition site in [D-(3-13C)Ala8]- and DL-[3-13C]Ala7-phospholipase A2, while these proteins still possess considerable enzymatic activity toward monomeric substrates. In contrast, however, kinetic experiments using monomolecular surface films, allowing a continuous change in surface density of the substrate molecules, revealed that [D-(3-13C)Ala8]- and DL-[3-13C]Ala7-phospholipase A2 at low surface pressure possess about 60 and 30% of the interface activity of native phospholipase A2, respectively. These results therefore suggest that the modified phospholipases A2 do possess an interface recognition site although less powerful as compared to that of the native enzyme, enabling the estimation of the surface density of micellar short chain lecithins.

Phospholipase C from Bacillus cereus. Action on some artificial lecithins.

The hydrolysis by phospholipase C from B. cereus of several lecithins of different fatty acyl chain length was examined. The enzyme showed significant activity towards mono-molecularly dispersed short chain lecithins and the reaction obeyed normal Michaelis-Menten kinetics. Rate vs. substrate concentration curves obtained with dihexanoyl-, diheptanoyl- and dioctanoyllecithins showed marked discontinuities in the region of the known critical micelle concentrations for these substrates and distinctly higher rates were obtained just above these levels. Using these three lecithins at levels below their respective critical micelle concentrations, rate increases were noted if the reactions were allowed to proceed to a sufficiently great extent. The presence of deoxycholate in the reaction system had little or no effect on the rate of enzyme-catalysed hydrolysis of lecithins of fatty acyl chain length less than or equal to Cbeta, but for fatty acyl chain lengths greater than C10, significant rate increases occurred. The pH profile for the enzyme activity was also examined.

Properties of Liposomal Membranes Composed of Short-Chain Lecithins

Permeability properties of multilamellar liposomes prepared from synthetic, saturated short-chain (C8, C10, C12) lecithins (3-sn-phosphatidyl choline) were studied. Dioctanoyllecithin, didecanoyllecithin, and dilauroyllecithin form "stable" bilayers which are practically impermeable to glucose when prepared with proportions of more than 1.2, 0.75, and 0.6 of cholesterol (molar ratio to phospholipid), respectively. Dioctanoyllecithin liposomes were rather leaky above 30 degrees, even when a proportion of 1.5 of cholesterol (molar ratio to lecithin) was incorporated. Judging from their sensitivities to temperature, Triton X-100, serum albumin, and other reagents, short-chain lecithin bilayers show characteristic properties, different from those of both saturated long-chain lecithins (dipalmitoyllecithin and dimyristoyllecithin) and lecithins having unsaturated fatty acid residues (egg lecithin and dioleoyllecithin).

Studies on lysophospholipases, v. the action of lysolecithin-hydrolyzing enzymes on lecithins and 1-acyl lysolecithins with varying fatty acid chain-length

The activity of two purified lysolecithin-hydrolyzing enzymes on homologous series of synthetic lecithins containing two identical fatty acyl chains and of 1-acyl-lysolecithins has been measured as a function of substrate concentration. In general, enzymatic activity toward lecithins decreased with increasing chain length. Maximal hydrolysis rates for the lysolecithin series were measured with 1-dodecanoyllysolecithin. In this series increased affinities for substrates with increasing acyl-chain length was noticed. In the substrate concentration versus enzymatic velocity curves no breaks were observed at the critical micelle concentration of the various substrates. The initial site of attack during hydrolysis of short-chain lecithins was determined using 1-octanoyl-2-pentanoyl-lecithin, 1-hexanoyl-2-hexyllecithin and 1-hexyl-2-hexanoyllecithin. Both enzymes exhibited a pronounced preference for hydrolysis of the acyl ester bond at the 1-position. Especially the enzyme from beef pancreas seems to be suitable for the enzymatic preparation of 2-acyl lysolecithins from the corresponding short-chain lecithins.

Microcalorimetric study of phospholipid binding to human apo-HDL

The binding of lysolecithin and synthetic short-chain lecithins: di-caproyl, di-lauroyl and di-myristoyl lecithins to a human apo-high density lipoprotein (apo-HDL) was followed by microcalorimetry. Complex formation was checked by ultracentrifugal flotation. The binding reaction was very rapid and strongly exothermal. The apparent binding enthalpy ΔH B together with the complex composition were computed from the binding curves. Both quantities were of the same order of magnitude for lysolecithin and for the shorter chain lecithins while the binding of di-myristoyl lecithin was characterized by a more highly exothermal reaction. The structure of the lipid phase strongly influences the enthalpy change. In the case of lysolecithin and of the shorter chain lecithins; which form micellar structures in water, the enthalpy change is mainly due to apoprotein-phospholipid complex formation. The disrupture of the myelin figures formed by the di-myristoyl lecithin accounts for the complementary heat effect. The phospholipid composition of the complexes isolated by ultracentrifugal flotation was lower than that determined by microcalorimetry, due to the presence of high salt concentrations in the ultracentrifuge.

Physical chemical studies of short-chain lecithin homologues. 3. Phase separation and light scattering studies on aqueous dioctanoyllecithin solutions.

Abstract Some phase separation phenomena in aqueous dioctanoyllecithin solutions including the effects of NaCl and Lil on the phase diagram are reported. At low electrolyte concentrations (below 0.2 M) both salts cause the upper consolute temperatures (u.c.t) to decrease, probably due to a decrease of electrostatic attraction between the lipid molecules. At higher salt concentrations the effect of Lil continues in the same direction (salting-in) but NaCl leads to an increase of the u.c.t. (salting-out). Micellar weight determinations could be performed at room temperature in homogeneous lecithin solutions containing 0.2 M Lil. An attempt was made to interpret the light scattering data with the help of the open association model (equal association constants for aggregation steps beyond a certain step) and the Flory-Huegins type of thermodynamic nonideality. The angular dependence of the light scattering points to very large and extended micelles. The radii of gyration are approximately proportional to the square root of the micellar weights.

Physical chemical studies of short-chain lecithin homologues. II: Micellar weights of dihexanoyl- and diheptanoyllecithin

The micellar weights of dihexanoyl- and diheptanoyllecithin in aqueous solutions are calculated from light scattering and ultracentrifugation data. A monomer-micelle assocation model is used and corrections for the thermodynamic nonideality, on the basis of rigid noninteracting particles, are applied. A few experiments on the influence of high NaCI concentrations (up to 3 M) are described. Dihexanoyllecithin forms micelles with micellar weight of 15 000 to 20 000 and with rather narrow weight distributions. Diheptanoyllecithin micelles however, have broad size distributions with micellar weights of 20 000 up to about 100 000 in the concentration range studied. Micelles are assumed to be spherical or to have sphero-cylindrical shapes depending on the molecular weights. Two models are used: (1) a compact structure, where no attention is paid to the hydrocarbon-water contact (2) micelles with as little hydrocarbon-water contact as possible.

Physical chemical studies of short-chain lecithin homologues. I.: Influence of the chain length of the fatty acid ester and of electrolytes on the critical micelle concentration

The critical micelle concentration (CMC) of four synthetic phosphatidylcholines (containing two hexanoyl, heptanoyl, octanoyl or nonanoyl residues respectively) in aqueous solutions have been determined by surface tension measurements. The dependence of the CMC on the chain length is discussed on the basis of the mass action model for micelle formation. For the three higher homologues a contribution of 1.08 kT per CH 2 group to the standard free energy of micellisation is found. The change in this free energy in going from the dihexanoyl- to the diheptanoyllecithin is somewhat larger (1.2 kT per CH 2 group). The influence of high concentrations (several moles per liter) of simple electrolytes on the CMC is interpreted as a salting-out of nonpolar solutes in water. Contrary to expectations the effects of NaCl and Lil on the CMC of dioctanoyllecithin are not additive.